INTRODUCTION:

In the ever-advancing landscape of medical science, pulmonary arterial hypertension (PAH) persistently remains a formidable adversary, presenting a myriad of challenges in diagnosis, treatment, and patient prognosis. Despite the significant progress made in understanding and treating various diseases, PAH's intricate nature renders it a daunting medical condition with often disheartening prognostic outcomes.

Empirical data underscores the pressing nature of this issue. The incidence rate of pulmonary hypertension stands at an estimated 4–10 cases per million children annually. When assessed on a broader scale, prevalence rates indicate a range of 20–40 cases per million in Europe and 26–33 per million children in the United States^1-4. Alarmingly, despite the indisputable severity and significant mortality associated with this disease, many countries lack a comprehensive registry for pulmonary hypertension. This oversight becomes even more distressing when considering that approximately 30% of children with non-amendable cardiac defects succumb to complications arising in the pulmonary vessels⁵.

Given these statistics, there's an urgent call for an aggressive approach to both the diagnosis and management of PAH. Right heart catheterization, despite being the current gold standard for diagnosing PAH, is an invasive technique fraught with potential complications. Other non-invasive modalities such as echocardiography, magnetic resonance tomography (MRT) of the heart, and computer tomography (CT) of the chest, while beneficial, come with their own sets of limitations^6-10.

In this light, the identification and understanding of PAH biomarkers have emerged as a promising avenue. Early detection through these biomarkers could revolutionize the way PAH is diagnosed and treated, thereby ameliorating patient outcomes. One such biomarker that has recently garnered significant attention is serotonin. Serotonin plays a quintessential role in regulating vascular tone and endothelial cell growth. The implications of serotonin on the developing pulmonary arteries in children can be profound, influencing long-term health outcomes. Emerging research suggests that altered serotonin metabolism might be intricately linked with vascular wall remodeling processes, a key factor in the development of pulmonary hypertension. Advancing our understanding of serotonin's role in PAH could pave the way for innovative diagnostic and therapeutic strategies ^11-19.

MATERIALS AND METHODS:

Study design – cohort study. The study was authorized by ethic committee of Karaganda Medical University No. 37 dd. 29.03.2022. Parents of all patients, involved into the study, signed the informed consent for the study. The selection of participants was carried out in the Center of cardiosurgery of Karaganda city (Kazakhstan) in the period from April 2022 till March 2023. Children were divided into three groups. I group – children with CHDs complicated with PAH, before the surgical correction of cardiac defect, II group – children with CHDs complicated with PAH, after the surgical correction of cardiac defect, III – control group (healthy children without CHD and PAH). Criteria for inclusion into I, II groups were the following: presence of detected CHD in children, pulmonary hypertension, absence of infectious complications, age of 0 to 7, consent of parents/legal representatives of children for participation in the study. Criteria for inclusion into III group: absence of severe somatic pathology, including CHD and PAH, absence of active infectious and inflammatory processes, age of 0 to 7, consent of parents/legal representatives of children for participation in the study.

Laboratory methods:

The following values were defined: amount of blood platelets and their characteristics (mean platelet volume, platelet distribution width, plateletcrit), serotonin concentration in blood serum and platelets, serotonin metabolite - 5-hydroxyindoleacetic acid (5-HIAA) in blood plasma, urine and platelets; serotonin transporter SERT in platelets and level of 5-НТ2А receptors to serotonin in platelets. The whole blood with citrate was used for extraction of platelets and EDTA-plasma to get the platelet poor plasma (Platelet Poor Plasma), and urine. Biological material (blood, urine) were collected before and after the surgical treatment.

Hematological studies:

Platelet count and platelet index (mean platelet volume, variation coefficient of platelet volume, plateletcrit) were estimated in the whole blood, using Hematology analyzer Mindray 3200. Platelets were extracted from citrated plasma. To get the blood plasma enriched with platelets (PET), samples were centrifuged for 5 minutes at room temperature at 200 х g. To get platelets, the platelet sediment was formed by adding 800 mcl of physiological solution to 200mcl PET and centrifuging (4500 х g, 10minutes at temperature of 4°С). Supernatant liquid was eliminated. 200mcl of distilled water were added to platelet sediment and vortex-mixed. This suspension was stored frozen at temperature minus 40°С. Before the assay, frozen sample was singly defrosted and frozen for better membranes destruction. For assay 100mcl of suspension were used. Values in platelets were recalculated for 10⁹platelets.

Immunoassay analysis:

To estimate 5-HIAA, SERT, 5-НТ2А, IFA assay sets were used, produced by Cloud-Clone Corp. Concentration in urine was recalculated for creatinine level to standardize the dilution effect of urine.

Instrumental survey:

In all the children data of echo-cardiography before and after the surgical treatment were examined.

Statistic methods:

Normal quantitative indices were described with mean (М) and standard deviations (СО). To compare 3 groups, on-way ANOVA was used (watch the Tables), for multiple intragroup comparison – multiple comparison test of Holm-Sidak.

RESULT:

The study encompassed a representative sample of 25 children, manifesting a mild phenotype of PAH. The ages of these children were demographically diverse, extending from 13 days to 6 years, with a mean age of approximately 16months. Within this population, a notable 40% presented with a ventricular septal defect (VSD), an identical percentage evidenced an atrial septal defect (ASD), while a smaller proportion, 4%, were diagnosed with patent ductus arteriosus (PDA). The remaining 16% exhibited a combined morphological cardiac anomaly.

Moreover, within a subgroup of five children, who ranged from 1 to 15 months of age with a median age of 5.6 months, a severe phenotype of PAH was scrutinized. This subgroup was characterized by the presence of profound combined cardiac defects, encompassing conditions such as double outlet right ventricle in conjunction with VSD and common arterial trunk coupled with ASD and VSD anomalies. Within this subset, 20% were diagnosed with second-grade PAH, while a significant majority, 80%, suffered from third-grade PAH. This condition was not delineated along gender lines, as both male and female patients were nearly equally represented.

Pertinent to the hemodynamic parameters assessed, the ratio of pulmonary to systemic blood flow (Qp/Qs) invariably exceeded unity across all patients, underscoring the existence of pulmonary hypertension. In a substantial 62% of this cohort, this ratio surpassed 2, indicative of a non-reversible progression of PAH. An alarming revelation was that in two patients, the tricuspid annular plane systolic excursion (TAPSE) values were significantly diminished (measuring 10 mm and 7mm, respectively), a clinical indicator of systolic dysfunction of the right ventricle. This metric was found to exhibit a correlative relationship with the left ventricular ejection fraction.

A further examination revealed that a majority, constituting 83% of patients, presented with an enlargement of the pulmonary artery, and in a fraction of these cases, this pathology was accompanied by dilation of the left cardiac chambers. Additionally, an interatrial septal dysfunction was identified in three patients, and this was invariably associated with an augmentation of the mean pressure gradient, a heightened incidence of tricuspid regurgitation, and dilatation of the right cardiac anatomy.

The clinical manifestations of cardiac insufficiency, as stratified across functional classes I to III, exhibited a direct correlation with the severity of PAH across the studied individuals. Interestingly, the platelet indices (table 1), particularly the mean platelet volume, remained consistent pre- and post-surgical intervention, with the exception that the preoperative mean platelet volume significantly deviated from control values, normalizing shortly after surgical intervention. However, the coefficient of variation of platelet volume and plateletcrit remained unaltered, implying resilience to the pathophysiological impact of PAH.

Table 1: Data on platelet counts and platelet indices.

Group I	Group II	Group III	ANOVA
Platelets count (103 cells/µl)	323.08± 102,33	370.50± 119.31	301.94± 68.08	0.08
MPV (%)	7.50±0.62	7.71±0.53	8.09±0.78	0.03
PDW (fl)	15.91±0.54	15.71±0.48	14.94±2.50	0.12
PCT (%)	0.23±0.07	0.27±0.07	0.24±0.05	0.11

In examining the serotonin concentration within platelets, no significant fluctuation was noted pre- and post-corrective surgery for CHD within the mild PAH patient group. Contrastingly, in severe PAH patients, a notable 20% reduction in serotonin levels post-surgery was observed, suggesting a pivotal role of serotonin in the pathogenesis of irreversible forms of PAH, the remodeling of pulmonary vasculature, and the resultant augmentative strain on the right ventricle.

The urinary assay for 5-HIAA (table 2) emerged as the most sensitive indicator of serotonin metabolism within this patient population. In the CHD-affected patients, the excretion of 5-HIAA was found to be exponentially elevated, being twentyfold higher than control values, which notably reduced by 2.5-fold following surgical intervention, albeit the statistical significance was diminished due to the limited urine sample size in the control cohort.

Table 2. Concentration of 5-HIAA in plasma, platelets and urine.

Group I	Group II	Group III	ANOVA
5-HIAA in plasma (ng/ml)	8.18±1.70	7.75±1.18	7.55±1.67	0.46
5-HIAA in platelets (ng/10⁹)	12.71± 4.09	10.99±4.73	10.63±4.95	0.23
5-HIAA in urine (ng/creatinine mmol)	16.50± 33.06	6.28±10.40	0.81±0.65	0.6

Subsequent regression analysis elucidated a negative correlation (R=-0.59, p<0.05) between plasma levels of 5-HIAA and the estimated mean pulmonary arterial pressure (EMPAP), thereby endorsing plasma 5-HIAA as a potential biomarker for EMPAP (figure 1).

Figure 1: Regression analysis 5-HIAA in plasma and EMPAP

The subsequent phase of the investigation involved the identification of serotonin receptors and the serotonin transporter (SERT) in platelets (table 3). A remarkable and statistically significant elevation of SERT expression was observed in the platelets of children with CHD, which then significantly decreased post-surgical correction (p=0.03), reinforcing the association between the serotonin signaling system and the evolution of PAH.

Table 3: SERT and serotonin receptors in platelets

Group I	Group II	Group III	ANOVA
SERT on platelets (pg/109)	347.35± 87.27	284.66± 55.29	295.00± 76.23	0.027
5-НТ2А onplatelets (pg/109)	504.05± 382.44	519.26± 395.31	522.09± 325.24	0.98

Correlation analysis (table 4) further explicated the intricate relationship within the serotonin metabolic cascade. Notably, significant correlations were established between platelet SERT expression and platelet 5-HIAA levels (R=0.74, p<0.001), and between platelet SERT expression and platelet serotonin receptor density (R=0.9, p<0.001).

Table 4: Correlation analysis between SERT, 5-HT2A, 5HIAA

5-HIAA	5-HIAA5-HIAA	SERT	5-HT2A
Plasma	Urine	Platelets
5-HIAA	1,00
Plasma
5-HIAA	-0,007	1,00
Urine
5-HIAA	0,17	0,00	1,00
Platelets
SERT 0,06	-0,19	0,74	1,00
5-HT2A 0,18	-0,19	0,68	0,90	1,00

DISCUSSION:

Our research aligns with multiple investigations that underscore the pivotal role of serotonin in PAH pathophysiology, drawing attention to its profound influence on vascular smooth muscle cells.

Serotonin, a potent neurotransmitter, demonstrates both vasoconstrictive and mitogenic effects on these cells. Detailed experimental evaluations have unveiled its ability to stimulate the mitotic activities of the smooth muscle cells within pulmonary arteries. Significantly, animal-based studies conducted in Kazan have presented intriguing findings; a discernible correlation was observed between the concentration of the serotonin metabolite, 5-HIAA, in urine and the severity of PAH²⁰. Further affirming these findings, Mustafin et al.'s research on children with CHDs manifested with PH unveiled an evident elevation in the 5-HIAA levels in patients diagnosed with PH²¹.

Our investigative endeavors corroborate the aforementioned conclusions. We observed that the most pronounced alterations in the serotonin metabolite occurred in urine. Furthermore, the 5-HIAA concentration in the urine of patients with CHD coexisting with PAH was substantially elevated when juxtaposed with the control cohort. Delving into clinical studies, Hervé et al. postulated an augmentation in plasma serotonin concentrations in PAH patients, pinpointing a correlation with right ventricular pressure. Analogously, in our patient cohort with PAH, the serotonin levels were markedly higher than those in the control group.

Adding another dimension to this complex interplay, Chan &Loscalzo unveiled the integral role of serotonin transporter (SERT) in modulating the smooth muscle cells of pulmonary vessels, thereby influencing PAH progression²². In line with these insights, our 2020 investigations revealed that the SERT concentrations in children diagnosed with PAH were considerably elevated compared to their non-PAH counterparts. Although our findings resonate with prior data, it's imperative to note that the extent of SERT elevation in PAH-afflicted children isn't overwhelmingly significant²³.

The cumulative data underscore a palpable association between serotonin metabolism and the onset and progression of PAH. This accentuates a critical juncture in our comprehension of the intricate dynamics governing the serotonin system and its subsequent ramifications on the cardiovascular apparatus. While our understanding has burgeoned over the years, substantial lacunae exist regarding the precise mechanisms delineating the synergistic interactions of the serotonin system with both the cardiovascular matrix and pulmonary hypertension^24-26.

To bridge these knowledge gaps, future research endeavors must possess a laser-focus on a more profound exploration of this system. By doing so, we can unlock its latent potential, heralding the advent of novel diagnostic techniques, therapeutic interventions, and early biomarker identification for PAH.

To sum up, our research accentuates the imperativeness of a nuanced examination of the serotonin system and its consequential effects on PAH pathogenesis. Amplifying our reservoir of knowledge in this domain can potentially pave the way for tailor-made diagnostic, therapeutic, and preventive strategies. This, in turn, could enhance the quality of life of afflicted patients, while concurrently mitigating disease-associated complications.

CONCLUSION:

The exploration of PAH in children with CHDs is a critical realm of scientific inquiry that holds the potential to significantly improve patient outcomes. This recent study sheds invaluable light on the complex mechanisms governing PAH in this vulnerable patient population, underscoring the profound importance of delving deeper into the intricate pathways of this condition.

The identification of potential biomarkers, such as urinary and plasma 5-HIAA levels, is yet another significant finding of this study. Biomarkers play a pivotal role in modern medicine, offering a tangible, quantifiable measure of disease presence, severity, and progression. In the case of PAH in children with CHD, the ability to monitor and measure these biomarkers can guide treatment decisions and allow for the early identification of potential complications. Beyond mere diagnostics, these biomarkers might provide insights into the pathogenesis of the condition itself, opening the door for innovative therapeutic strategies.

Unraveling the precise mechanisms underlying PAH in children with CHD is not a straightforward task. The condition's multifactorial nature requires a multidisciplinary approach, combining insights from cardiology, molecular biology, pharmacology, and even neurobiology. The potential rewards of such investigations are immense. By understanding the very essence of the disease process, the medical community can identify therapeutic targets that can halt or even reverse the progression of PAH.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

This research has been funded by the Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan (Grant No. AP14871767)

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Received on 09.11.2023 Modified on 18.12.2023

Research J. Pharm. and Tech 2024; 17(7):3073-3078.

DOI: 10.52711/0974-360X.2024.00481