Membrane serotonin transporter as a Biomarker of Pulmonary arterial hypertension in children with Congenital Heart Defect

Mindubaуeva F.¹, Niyazova Y.¹, Nigmatullina R.², Sadykova D.², Akhmaltdinova L.¹, Salikhova Y.¹, Kadyrova I.¹, Akhmetova M.¹, Sabirova D.²

¹“Karaganda Medical University”, NCJSC

²Kazan State Medical University, Kazan, Russia.

*Corresponding Author E-mail: 7554422@mail.ru, yuliya_niyazova99@mail.ru

ABSTRACT:

18 young children (aged from 1 month old to 2 years old) with congenital heart disease were observed. During observation they were divided into 2 groups. Group I - 8 children with CHD, not complicated by PAH. Group II 11 children with CHD, complicated by PAH. The content of serotonin and its transporter in platelets, the amount of serotonin in serum were determined. Serotonin content was determined by enzyme-linked immunosorbent assay. The study was performed using the Serotonin ELISA diagnostic kit, IBL Hamburg on the Evolis automated robotic station (BioRad). To determine the Sodium-Dependent serotonin transporter (SLC6A4) there was used the method of quantitative sandwich ELISA of Cusabio production. The main initiator of the development of PAH is development of endothelial dysfunction of the pulmonary vessels. Since the cause of this dysfunction is associated with functional disorders of the serotonergic system, our task was to determine the content of SERT and serotonin in platelets in the studied groups. We suggest that the serotonin transporter (SERT), which is responsible for the proliferation of the pulmonary artery smooth-muscle cells (PASMC), can be used as a biomarker for predicting and diagnosing PAH in children with CHD. The need for early biomarkers in the diagnosis of PAH in children is claimed by scientists from many countries of the world [1,2]. The results of our studies showed a significant increase in the concentration of SERT in platelets in group II, compared with group I more than several times. The serotonin content in platelets in children of group II tended to decrease as compared with group I. Perhaps, an excessive increase in transporters in platelets is sufficient in itself for the induction of PASMC hyperplasia and the subsequent development of PAH. It is necessary to continue research in this direction, which will help to reveal and understand the molecular mechanisms by which SERT regulates the proliferation of PASMC and the role of serotonin and its transporter in the development of PAH in children.

KEYWORDS: CHD, PAH, children, serotonin, SERT.

INTRODUCTION:

Pulmonary arterial hypertension (PAH) in children is considered to be a complex and multifactorial disease, the survival of which depends on timely diagnosis at an early stage of the disease. The issue of PAH associated with congenital heart disease (CHD) is particularly acute. In this connection, the search for biomarkers is needed to predict the development of PAH in young children with CHD.

Pathological changes in PAH are characterized by intimal hyperplasia, media hypertrophy, adventitious proliferation, obliteration of small arteries, and vasculitis, which are the reasons for the rapid lethal outcome [3]. The exact mechanisms responsible for the pathogenesis of pulmonary hypertension (PH) have not yet been elucidated, however, the cause of vascular remodeling is associated with the dysfunction of the serotonergic system, where control of cell proliferation is lost. We suggest that the serotonin carrier (SERT), which is responsible for the proliferation of the pulmonary artery smooth-muscle cells (PASMC), can be used as a biomarker for predicting and diagnosing PAH in children with CHD.

MATERIALS AND METHODS:

The serotonin content in platelets, serum, and its transporter concentration in platelets were determined. To determine serotonin system parameters, blood was taken from the children in fasting state into two tubes, one for serum, the other for platelets and aggregation with sodium citrate (for serotonin and transporter). Prior to the study, the serum was stored at a temperature of -70 degrees Celsius.

To obtain platelet-rich plasma (PRP), the samples were centrifuged for 5 minutes at room temperature at 200 x g. The supernatant was transferred to another tube and platelets were counted on a Mindray 3200 hematology analyzer. To obtain platelets, the pellets pellet was obtained by adding 800μl of saline to 200μl of PRP and centrifuged (4500 x g, 10 minutes at 4°C). The supernatant was removed. 200μl of distilled water was added to the platelet pellet and vortexed thoroughly. The resulting suspension was stored frozen at a temperature of minus 40°C. The frozen sample was subjected to single thawing and freezing prior to analysis, 100μl of the suspension was used to better disrupt the membranes for analysis.

Determination of serotonin in platelets and serum was carried out separately. Serotonin content was determined by enzyme-linked immunosorbent assay. The study was performed using the Serotonin ELISA, IBL Hamburg diagnostic kit on the Evolis automated robotic station (BioRad).

To determine the Sodium-Dependent serotonin transporter (SLC6A4) there was used the method of quantitative sandwich ELISA of Cusabio production. Monoclonal antibodies to SLC6A4 were pre-coated on a microplate. Standards and samples were pipetted into the wells according to the manufacturer’s instructions and bound to immobilized antibodies. After the removal of unbound substances, biotin-conjugated antibodies specific for SLC6A4 were added to the reaction wells. After subsequent washing, avidin-enzyme reagent and substrate solution were added to visualize the reaction. The analysis was carried out on the Evolis robotic immunoassay system, the calculation of the results was carried out by the embedded software. In order to express the content of Sodium-Dependent serotonin transporter in platelets, recalculation was made of the number of platelets in the sample obtained at the platelet extraction stage and expressed as pg/10⁹ platelets.

Statistical processing of the material was carried out using the standard application of Statistica application programs for a personal computer. The significance of differences was evaluated using the Mann – Whitney U-test. Results are presented as M ± σ.

RESULTS AND DISCUSSION:

Among congenital heart defects in group I, 67% of children had VSD. In group II, VSD was 64%, in 36% of those studied, it was combined with other heart defects.

Indicators of serotonin system in children in groups are presented in the table (Table 1). There was a marked increase in the concentration of SERT in the group of children with CHD and PAH compared with the group of children with CHD.

The amount of serotonin in the serum of children in group II with PAH, compared with the group of children with CHD, tended to increase. Serotonin levels in platelets in the group of children with CHD complicated by PAH tended to decrease. However, in 30% of cases of children of the group II, the SERT was 575.5±258.4 pg/10⁹ and there was a tendency to an increase in the serotonin content in platelets. The number of platelets in the blood of children of both groups was within the age norm.

It is known that the serotonin transporter (SERT), a sodium-dependent intracellular protein, product of SLC6A4 gene, performs the function of the reuptake and transfer of serotonin from the synaptic cleft into presynaptic terminals, thereby reducing the effects of serotonin on receptors in the postsynaptic membrane [4, 5]. SERT is present on the plasma membranes of platelets [6], cardiomyocytes [7], pulmonary endothelium [8] and placental epithelium [9], where it participates in systemic serotonin homeostasis. SERT activation and inactivation is regulated through the activity of protein kinase G [10].

A serotonin transporter is responsible for the proliferation of pulmonary artery smooth-muscle cells (PASMC), stimulated by serotonin (5-HT). This is accomplished by active transport of 5-HT intracellularly, followed by tyrosine phosphorylation of the GTPase-activating protein, formation of reactive oxygen intermediate and Erk MAPK signaling [11]. SERT interacts with 5-HT receptors (5-HTR) in the production of PASMC 5-HT-induced proliferation via the Rho, Erk and PI3K/Akt G protein [12]. RhoA serotonylation after SERT-mediated cellular internalization of serotonin in platelets was described by a group of scientists led by Walther DJ [13]. The activity of RhoA and Rho kinase is increased with idiopathic PAH (iPAH) due to increased serotonylation of RhoA, and this may precipitate platelet activation [14]. Serotonin-induced fibrosis may also play a role in the formation of PAH. Serotonin can activate pulmonary arterial fibroblasts and promote fibrosis of adventitia through signaling via the TGFb1 / Smad327 pathway [15] and, at PASMC, via NADPH oxidase (Nox1) [16].

In our studies, we revealed a tendency to a decrease in the serotonin content in platelets in children with congenital heart disease, complicated by pulmonary arterial hypertension. Probably, its reuptake by a transporter is broken. But the concentration of SERT in platelets significantly increased in children with PAH.

The mechanisms of SERT involvement in the pathogenesis of pulmonary hypertension were mainly studied experimentally [17,18]. It was revealed that SERT, along with other growth factors: platelet derived growth factor (PDGF), fibroblast growth factor (FGF) and epidermal growth factor (EGF), have been found to affect PASMC proliferation in cattle [18]. The results of the study, using SM22-SERT mice (transgenic mice with overexpression of SERT occurring selectively in smooth muscle cells), indicate that overexpression of the vascular SERT protein alone is sufficient to induce PASMC hyperplasia and subsequent PH, despite lack of associated changes in the bioavailability of 5-HT and other stimuli, such as hypoxia, which increases the likelihood that, in addition to its role in 5-HT signaling, the SERT may also be involved in the co-regulation of proliferation by other mitogens PASMC [17].

Human studies are sporadic. Castro E.C. with a group of scientists studied the expression of SERT in newborns with persistent pulmonary hypertension using lung sections. They have identified the ability of SERT to accumulate in the vessels and endothelium of the lungs. In their results, they showed that a decrease in SERT activity leads to an increase in serum serotonin level [19].

If the serotonin level in serum increases, then the serotonin content in platelets should decrease, which was noted in our studies and in studies conducted by Brenner B. et al., who showed that the concentration of 5HT in platelets in hypertensive patients was 33% lower compared to with platelets in normotonics. The concentration of 5HT in hypertensive blood was increased by 33% [20]. However, the preliminary action of serotonin on platelets isolated from normotensive blood samples leads to a decrease in the level of SERT, which is typical for platelets isolated from hypertensive blood.

One of the reasons for the reduction of serotonin in platelets may be due to a violation of the mechanisms of serotonin reuptake by the transporter. But the content of the SERT in our case is increased in children with PAH.

We suggest that an increase in SERT in children with PAH is associated with a decrease in their activity due to conformational processes, a carrier of serotonin in platelets. The transport of 5-HT occurs via an alternating access mechanism, where the binding of a substrate with the length of Na + and Cl - ions transported together causes a conformational change in SERT from the conformation facing outward to the conformation facing inward [21,22]. The issues of the structure and regulation of the 5HT transporter have been little disclosed to date [23]. It was revealed that the site of capture of 5-HT human platelets is identical to the carrier of 5-HT human brain and approximately 92% homologous to the rat protein [24]. There was an attempt to clone serotonin transporters and its expression in the experiment [25]. It was shown that the activity of serotonin transport increases 2.4 times in the cells of the placental human choriocarcinoma JAR after treatment with cholera toxin or forskolin. Perhaps this is the result of an increase in the density of the serotonin transporter protein on the cell membrane as a result of elevated steady state serotonin transporter mRNA levels [26].

CONCLUSION:

Thus, determination of the concentration of SERT in platelets can serve as a PAH biomarker in children with congenital heart defects. Perhaps, an excessive increase in transporters in platelets is in itself sufficient for the induction of PASMC hyperplasia and the subsequent development of PAH. The results of our work point to the need for further research, which will enable us to understand the molecular mechanisms by which SERT regulates the proliferation of PASMC and the role of serotonin and its transporter in the development of PAH in children.

CONFIRMATION:

The research is funded by the MES RK (Grant No. AP05136034).

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

REFERENCES:

1. Kelley L. Biomarkers for pediatric pulmonary arterial hypertension- a call to collaborate Frontiers in Pediatrics. Pediatric Pulmonology. 2014; 2. Article 7. doi: 10.3389 fped.2014.00007.

2. Marius M. Difinitions and diagnosis of Pulmonary hypertension . Journal of the American College of Cardiology. 2013; 62: 25.

3. Avdeev S.N. Pulmonary hypertension.GEOTAR- Media. 2015; 416.

4. Nebigil, C.G., Pharm D., Maroteaux L. Functional consequence of serotonin 5-HT2B receptor signaling in heart. Role of mitochondria in transition between hytertrophy and heart failure. Circulation. 2003; 108: 902-908.

5. Cote, F. Disruption of the nonneuronal tph 1 gene demonstrates the importance of peripheral serotonin in cardiac function/ F. Cote, E. Thevenot, C. Fligny. Proc. natl. acad. sci. USA. 2004;100: 13525-13530.

6. Blakely RD, Berson HE, Fremeau RT, Jr, Caron MC, Peek MM, Priace HK, Bradley CC. Cloning and expression of a functional the 5HT transporter from rat brain. Nature. 1991; 354: 66–70.

7. Hoffman BJ, Mezey E, Browstein MJ. Cloning of the 5HT transporter affected by antidepressants. Science. 1991;254:579–580.

8. Lesch KP, Bengel D, Heils A, Sabol SZ, Greenberg BD, Petri S, Benjamin J, Muller CR, Hamer DH, Murphy DL. Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. Science. 1996; 29:1527–1531.

9. Ramamoorthy S, Cool DR, Mahesh VB, Leibach FH, Melikian HE, Blakely RD, Ganapathy V. Regulation of the human serotonin transporter. Cholera toxin-induced stimulation of serotonin uptake in human placental choriocarcinoma cells is accompanied by increased serotonin transporter mRNA levels and serotonin transporter-specific ligand binding. J. Biol. Chem. 1993a ;268:21626–21631.

10. Rudnick G. Active transport of 5-hydroxytryptamine by plasma membrane vesicles isolated from human blood platelets. J. biol. chem. 1977; 252: 2170-2174.

11. Nigmatullina R.R., Matveeva V.L., Chibireva M.D. The influence of selective serotonin re-uptake inhibitor fluoxetine on inotropic function of myocardium in the ontogenesis of rats. Ross FiziolZhIm I M Sechenova. 2014; 100(3): 348-359.

12. GillisC.N., Pitt B.R. The fate of circulating amines within the pulmonary circulation. Annu rev physiol. 1982; 44: 269-281.

13. Balkovetz D.F., Tiruppathi C., Leibach F.H. Evidence for an imipramine-sensitive serotonin transporter in human placental brush-border membranes. J. biol. chem. 1989; 264: 2195-2198.

14. Zhu C.B., Hewlett W.A., Francis S.H. Stimulation of serotonin transport by the cyclic GMP phosphodiesterase-5 inhibitor sildenafil. Eur j. pharmacol. 2004; 1: 1-6.

15. Ren W., Watts S.W., FanburgB.L.Serotonin transporter interacts with the PDGF receptor in PDGF-BB-induced signaling and mitogenesis in pulmonary artery smooth muscle.Am J Physiol Lung Cell Mol Physiol. 2011; 300: 486–497.

16. Liu Y.,FanburgB.L. Serotonin-induced growth of pulmonary artery smooth muscle requires activation of phosphatidylinositol 3-kinase/serinethreonine protein kinase B/mammalian target of rapamycin/p70 ribosomal S6 kinase 1.Am J Respir Cell Mol Biol. 2006; 34: 182–191.

17. Walther D.J.,Peter J.U., Winter S. Serotonylation of small GTPases is a signal transduction pathway that triggers platelet alpha-granule release. Cell. 2003; 115: 851–862.

18. Guilluy C.,Eddahibi S., AgardC. RhoA and Rho kinase activation in human pulmonary hypertension: role of 5-HT signaling. Am J RespirCrit Care Med. 2009; 179: 1151–1158.

19. Chen C.,Han X., Fan F. Serotonin drives the activation of pulmonary artery adventitial fibroblasts and TGF-þƒ1/ Smad3-mediated fibrotic responses through 5-HT2A receptors. Mol Cell Biochem. 2014; 397: 267–276.

20. Hood K.Y., Montezano A.C., Harvey A.P. Nicotinamide adenine dinucleotide phosphate oxidase-mediated redox signaling and vascular remodeling by 16alpha-hydroxyestrone in human pulmonary artery cells: implications in pulmonary arterial hypertension. Hypertension. 2016; 68: 796–808.

21. Guignabert C.,Izikki M., Tu L.I. Transgenic mice overexpressing the 5-hydroxytryptamine transporter gene in smooth muscle develop pulmonary hypertension. Circ Res. 2006; 98: 1323–1330.

22. Lee S.L.,Wang W.W., Moore B.J. Dual effect of serotonin on growth of bovine pulmonary artery smooth muscle cells in culture. Circ Res. 1991; 68: 1362–1368.

23. Castro E. C. C.,Sen P., Parks W. T.,Langston C., Galambos C. The Role of Serotonin Transporter in Human Lung Development and in Neonatal Lung Disorders. Canadian Respiratory Journal. 2017. – 10 pages. - Article ID 9064046, https://doi.org/10.1155/2017/9064046.

24. Brenner B, Harney JT, Ahmed BA, Jeffus BC, Unal R, Mehta JL, Kilic F. Plasma serotonin level and the platelet serotonin transporter. J Neurochem. 2007;102:206–216

25. Broer S, Gether U. The solute carrier 6 family of transporters. Br J Pharmacol. 2012;167:256–278.

26. Kristensen AS, Andersen J, Jorgensen TN, Sorensen L, Eriksen J, Loland CJ, Stromgaard K, Gether U. SLC6 neurotransmitter transporters: structure, function, and regulation. Pharmacol Rev. 2011;63:585–640.

Received on 27.09.2019 Modified on 11.11.2019

Research J. Pharm. and Tech 2020; 13(5): 2435-2438.

DOI: 10.5958/0974-360X.2020.00436.9

Group	n	Serotonin in serum, ng\10⁹	Serotonin in platelets, ng\10⁹	SERT in platelets, pg/10⁹	*Number of platelets, 10⁹/l**
I CHD	8	195.9±70.7	373.4±190.7	394.6±136.2	304±97.8
II CHD+PAH	10	236.9±124.8	297.0±158.9	2968.4±2144.3*	336±106.3