Pro-regenerative effects of 5-hydroxytryptamine in cultured dermal fibroblasts and subcutaneous adipose tissue-derived mesenchymal stromal cells

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Background

Dermal fibroblasts (DFs) and subcutaneous adipose tissue-derived mesenchymal stromal cells (SAT-MSCs) play a critical role in the epithelial regeneration of damage- or age-related morphological defects and deficient structures. DFs and SAT-MSCs are recognized for their ability to proliferate and differentiate into skin cells to replace damaged or dead cells. In addition, they have been observed to promote cell regeneration and wound healing through autocrine and paracrine mechanisms [1]. Wound healing is associated with the rapid involvement of progenitor and subcutaneous adipose tissue-derived stem cells in regenerative processes. These cells migrate to injury sites and simultaneously differentiate into DFs, endothelial cells, and keratinocytes [2–6]. Furthermore, DFs and SAT-MSCs have been identified as the primary sources of extracellular matrix (ECM) proteins, which contribute to the maintenance of skin integrity and function. The interaction of ECM proteins and skin cells is intricately related to the maintenance of skin homeostasis and wound healing. The existing evidence suggests a regulatory role for DFs and SAT-MSCs in the inflammatory response to tissue damage. These cells modulate the phenotype of macrophages involved in the inflammation phase and promote angiogenesis by stimulating endothelial cell differentiation and cell migration. Furthermore, they contribute to the formation of granulation tissue, skin cells, and ECM production, thereby facilitating proliferation and remodeling [7]. Despite their promising potential for differentiation, migration, and paracrine effects to repair damaged tissue, the use of DFs and SAT-MSCs remains challenging because of the low cell engraftment rate at the transplantation site and, consequently, the low survival rate in suboptimal transplantation conditions. In recent practice, the use of various progenitor and stem cells for self-therapy has been replaced by an approach in which the body’s own stem cells are activated by extracellular factors such as vesicles, cytokines, etc. [8–14]. The role of numerous systemic humoral regulators, such as hormones and cytokines responsible for the intercellular communication, in particular biogenic amines with a wide range of biological activity, remains understudied.

Specifically, 5-hydroxytryptamine (5-HT, serotonin) is among the most extensively studied neurotransmitters, with a wide range of physiological functions extending beyond the central nervous system. These include the stimulation of cytokine and chemokine production, vasoconstriction, the regeneration of tissues and cells (fibroblasts, smooth muscle cells, and endothelial cells), proliferation and migration (eosinophils, mast cells, and dendritic cells), and the regulation of the immune system [15–17]. It has been established that 5-HT exerts its effects through binding to cell surface receptors, which are classified into seven families (5-HT1 to 5-HT7), with 14 different subtypes arising from structural diversity and modes of action. The effects of 5-HT on inflammatory cells are largely mediated by one or more of the following receptors: 5-НТ1А, 5-НТ2А, 5-HT3, 5-HT4, and 5-HT7 [18].

One study has identified the role of 5-HT as an important metabolic hormone that contributes to glucose homeostasis and obesity. A causal relationship exists between circulating 5-HT concentrations and metabolic diseases [19].

A mouse model of auxiliary partial orthotopic liver transplantation has shown that platelet-derived serotonin mediates liver regeneration after extensive hepatectomy and improves survival rates in mice. These observations were associated with the induction of 5-HT receptor (5-HTR2) expression after hepatectomy [20, 21]. The discovered effects of serotonin on increased liver tissue proliferation suggest its potential as a stimulation cofactor for the treatment of medical conditions characterized by reduced regenerative potential and compromised tissue healing. However, the available publications address only a limited amount of information concerning the regenerative and proliferative effects of serotonin on other tissues and cells. The effect of this hormone on skin regeneration has aroused particular interest and contributed to further research in this area.

Aim

The aim of this study was to examine the effect of serotonin on the in vitro regenerative potential (proliferation, migration, and cell death) of DFs and SAT-MSCs.

Methods

The study involved the experiments with primary cultures of DFs and SAT-MSCs obtained from Wistar rats using standard proved procedures, as documented in the protocols “Isolation, cultivation, and cryopreservation of the primary culture of human dermal fibroblasts” and “Isolation, cultivation, and cryopreservation of the primary culture of human subcutaneous adipose tissue-derived mesenchymal cells,” respectively [22].

The cell cultures were kept alive in a CO2 incubator (Binder CB150, Germany) with the culture medium being changed every three days (the medium was prepared for the experimental series) in the clean environment of a laminar flow microbiological safety cabinet (BMB-II-Laminar-C-1.8; Neoteric, Russia).

Upon achieving the desired mass, the cultured DFs and SAT-MSCs were divided into two series (a total of four series) of six biological replicates. The inoculum was transferred at 10,000 cells/dish into 3.5-cm Petri dishes. In 24 hours after the transfer, the standard culture media were replaced with those assigned to the experimental series: DFs cultured in the standard culture medium; SAT-MSCs cultured in the standard culture medium; DFs cultured in the standard culture medium with the addition of serotonin adipinate (LORR+K, Russia); SAT-MSCs cultured in the standard culture medium with the addition of serotonin adipinate. Serotonin adipinate was added to the medium in an amount of 10 mg/mL (6.6 μg per 1 mL of the culture medium). This particular concentration was empirically determined through a series of experiments in animal models to ensure its effectiveness, and it was subsequently recalculated to adjust the amount of culture media required for the in vivo experiment.

The statistical analysis of the obtained results was conducted using MS Excel, with hypotheses compared using the Mann–Whitney U-test and the Welch t-test via online calculators (https://www.statskingdom.com/170median_mann_whitney.html; https://www.statskingdom.com/150MeanT2uneq.html). Differences were considered statistically significant at p < 0.05.

The proliferation rate was calculated based on daily cell counts in 30 random visual fields in each dish using an Axio Vert.A1 microscope (Carl Zeiss, Germany) at ×50.

The migration rate of DFs and SAT-MSCs was determined using the scratch assay [23] in the standard medium and with serotonin, using ZEN Microscopy software (Carl Zeiss, Germany). The post-scratch Axio Vert.A1 images were captured 24 hours later to assess the recovery process of the defect. For an analysis and calculation of the mean values, the “culture wound” was observed in more than 10 visual fields for each series of cell cultures, each consisting of four biological replicates. Two observation approaches were used. The first approach involved measuring the “defect” reduction in micrometers from the wound edges to achieve the wound closure. The second approach focused on counting the cells that migrated beyond the “scratch” edges on both sides.

A quantitative analysis of apoptotic and/or necrotic cell death was performed by double-staining fluorescence microscopy (EVOS M7000 imaging system; Thermo Fisher Scientific, USA). The cells were stained with annexin (green fluorescence; signal with excitation wavelength 488 nm; emission wavelength 525 nm) and propidium iodide (PI; red fluorescence; excitation wavelength 535 nm; emission wavelength 615 nm). The live/dead cell assay was performed using an Annexin V-FITC/PI kit (ServiceBio, China) by examining 30 random visual fields in four Petri dishes per series to count various cell populations cells: apoptotic (annexin+PI–) and necrotic (annexin+PI+). In the present study, adherent cells in Petri dishes and washed incubation media on slides were stained with annexin and PI, which was followed by visual field microscopy.

Results

Cell Morphology by Phase-Contrast Microscopy

A comparison of the morphological characteristics of the DF and SAT-MSC cultures in the standard culture medium and the control series with serotonin showed no differences. The cultures were notable for a significant proportion of stellate, polygonal, and flattened cell shapes, which were consistent with the morphological patterns of fibroblast-like cells and SAT-MSCs, respectively. These cells were characterized by identifiable nuclei and nucleoli (two to three) and exhibited nonuniform cytoplasmic density and perinuclear grains in the presence of secretory activity.

Effect of Serotonin on Cell Proliferation

The DF cultures with and without serotonin showed differences in cell growth (see Fig. 1). The percentage of cells cultured in the medium with serotonin increased by 22% (p = 0.0003), 45% (p = 0.00004), 25% (p = 0.0003), 35% (p = 0.00003), and 13% (p = 0.005) on experimental days 1, 2, 3, 4, and 5, respectively. Furthermore, SAT-MSCs demonstrated an enhanced proliferative response to the addition of serotonin to the standard medium (p < 0.001). The percentage of SAT-MSCs cultured in the medium with serotonin increased by 5.4% (p = 0.3), 12.6% (p = 0.00002), 6.8% (p = 0.49), 25% (p = 0.0004), and 25.5% (p = 0.03) on experimental days 1, 2, 3, 4, and 5, respectively.

 

Fig. 1. Cell proliferation rates under serotonin adipinate exposure relative to control (n = 6), assessed by daily counting in 30 fields of view in experimental series: a, DF growth curve (n = 6); b, SAT-MSC growth curve (n = 6). DFs, dermal fibroblasts; SAT-MSCs, subcutaneous adipose tissue-derived mesenchymal stromal cells.

 

The time to reach a doubling of DFs was 31 hours for the control group, and 27 hours for the cells cultured in the medium with serotonin adipinate. In the SAT-MSC cultures, the doubling time was 38.2 hours in the control series and 35.7 hours in the series cultured with serotonin.

Effect of Serotonin on Cell Migration

The scratch migration assay demonstrated that the mean distance traveled by DFs incubated in the standard culture medium for 24 hours was 244.2 ± 13.0 μm (see Fig. 2). For SAT-MSCs growing in the standard medium, the respective value was 133.1 ± 23.0 µm.

 

Fig. 2. Assessment of DFs migration at 24 hours (µm:): a, control (n = 40); b, culture supplemented with serotonin (n = 40); original magnification ×50.

 

A conditional migration rate of DFs incubated with serotonin was slightly higher than for the standard medium, whereas SAT-MSCs cultures with serotonin exhibited a more significant increase. During the observed time period, the mean migration distance in the presence of serotonin was 248.9 ± 18.9 μm (p = 0.7) for DFs and 209.6 μm (p = 0.0001) for subcutaneous adipose tissue-derived cells.

The quantitative analysis of migrated cells (see Fig. 3) demonstrated an increase in the conditional migration rate for SAT-MSCs with serotonin, whereas the rate of DF migration remained similar to that observed in the control samples.

 

Fig. 3. Migration of DFs (n = 40) and SAT-MSCs (n = 40) at 24 hours, assesed by measuring the distance traveled by cells in serotonin-treated cultures compared with controls (n = 40). DFs, dermal fibroblasts; SAT-MSCs, subcutaneous adipose tissue-derived mesenchymal stromal cells.

 

Effect of Serotonin on Cell Death Pathways

The cell death pathways associated with apoptosis and/or necrosis were evaluated using double staining with annexin and propidium iodide (see Fig. 4). The series of dermal cells cultured in the standard culture medium (with four biological replicates each) was characterized by three to four apoptotic cells (annexin+PI–) and 22–24 necrotic cells (annexin+PI+) in 30 visual fields. The addition of serotonin to the culture medium resulted in an average of one to two cells positive for signs of apoptosis (annexin+PI–) (p = 0.006) and 17–20 necrotic cells (annexin+PI+) (p = 0.02) in 30 visual fields.

 

Fig. 4. Assessment of dermal fibroblasts cell death following serotonin exposure versus control, based on four biological replicates (30 fields of view each), using dual fluorescent staining with annexin (AN) and propidium iodide (PI): a, histogram of mean apoptosis/necrosis rates; b, composite image of adherent cells, combining phase-contrast microscopy of cells with two fluorescence images obtained at excitation/emission wavelengths of 488/525 nm for annexin (green) and 535/615 nm for propidium iodide (red-orange); c, composite image (as above) of suspended cells; original magnification ×200.

 

Discussion

The advancements in cell technologies have recently prompted a shift from artificial biomaterials to the modulation of regenerative processes that enhance the intrinsic healing mechanisms. Sadiq et al. investigated the effect of serotonin on primary DF cultures and neonatal epidermal keratinocytes. The authors observed an increased cell survival, a finding that was subsequently corroborated by a decrease in cell viability through the disruption of serotonin signaling by the treatment with ketanserin (a 5-HTR2A inhibitor) and fluoxetine [24]. Furthermore, a substantial body of research suggests that autologous platelets enhances skin wound healing [25], and they may function as serotonin reservoirs, which could potentially trigger the mechanism of cell adhesion and migration. The above factors and the involvement of dermal and subdermal layers in wound healing have contributed to the hypothesis that serotonin signaling can enhance the regenerative potential of both dermal progenitor cells and subcutaneous adipose tissue-derived cells, thereby promoting efficient wound healing. Accordingly, the findings of the present in vitro experiments demonstrate that cell cultures with serotonin exhibited enhanced survival rates and decreased cell doubling times compared with the control cultures. This observation, in addition to the study by Sadiq et al., suggests the implication of shared signaling mechanisms that regulate stem and progenitor cells within different tissues.

Naito et al. [26] and Kimura et al. [27] have previously attempted to elucidate the mechanisms of the regenerative effects of serotonin. These studies found that 5-HT stimulated the autocrine secretion of TNF-α from hepatocytes through 5-HT2B/Gq/phosphoinositide-specific phospholipase C/Ca2+, and TGF-α directly stimulates DNA synthesis and proliferation of hepatocytes (see Fig. 5). The mechanism of the proliferative effect of serotonin and the activation of migration has also been extensively investigated in hepatocellular carcinoma cells [28]. The activation of 5-HTR1D receptor has been demonstrated to trigger Tcf/Lef activation, which results in the accumulation of β-catenin and ultimately triggers cell proliferation and differentiation (see Fig. 6). Activation of 5-HTR1D receptor may also activate FOXO6 via the PI3K/AKT pathway, which, along with activation of the AKT/FOXO3α pathway through 5-HTR2B, promotes proliferation, colony formation, and migration of hepatocellular carcinoma cells. Furthermore, 5-HTR1D signaling has been observed to increase the expression of proteins associated with the Wnt/β-catenin pathway and target genes implicated in the over-proliferation of tumor cells, including β-catenin, survivin, C-myc, and cyclin D1. These pathways also activate the mTOR complex, thereby inhibiting autophagy, which is an additional factor contributing to the positive effect of serotonin on proliferation [18].

 

Fig. 5. Intracellular 5-HT receptor signaling via autocrine TGF-α–dependent stimulation of DNA synthesis and proliferation. 5-HT, 5-hydroxytryptamine/serotonine; 5-HTRs, 5-HT receptors; RTK, receptor tyrosine kinase; PLC, phospholipase C; PIP2, phosphatidylinositol 4,5-bisphosphate; DG, diacylglycerol; IP3, inositol 1,4,5-triphosphate; PI3K, phosphoinositide 3-kinase; ERK2, extracellular signal-regulated kinase 2; mTOR, mammalian target of rapamycin; TGF-α, transforming growth factor α.

 

Fig. 6. Cell signaling pathways activated by various 5-HT receptor subtypes in hepatocellular carcinoma. 5-HT, 5-hydroxytryptamine/serotonin; 5-HTRs, 5-HT receptors; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; АКТ, serine/threonine protein kinases (protein kinase B).

 

An analysis of the cell death pathways in the study [24] showed that serotonin had a significant antiapoptotic effect on fibroblast cultures, which also agreed with the effects observed for DFs and SAT-MSCs in the present study. This effect is evident from a decrease in apoptosis, which may elucidate one of the pathways (or mechanisms) through which serotonin facilitates cell growth, as evidenced in this study.

The immune cell adhesion and migration are critical for various functions, including extravasation, chemotaxis, phagocytosis, antigen presentation, and secretion of migrating cytokines and ECM. The activation of 5-HTR has the potential to directly or indirectly influence these mechanisms. It has been demonstrated that 5-HT exerts a chemotactic effect on eosinophils [29] and induces the in vitro 5-HT2A-dependent migration of human eosinophils and the in vivo movement and migration of mouse bone marrow-derived eosinophils in inflamed postcapillary venules [13]. According to the findings of this study, DFs cultured in the medium with serotonin demonstrated an increase in the conditional migration rates. A statistically significant and substantial increase in the conditional migration rate was documented for SAT-MSCs. John Jayakumar et al. [29] demonstrated that serotonin activation of 5-HTR played a significant role in adhesion, cytoskeletal remodeling, migration, and proliferation of vascular smooth muscle cells, mast cells, eosinophils, and dendritic cells. The 5-HT-induced migration of smooth muscle cells was demonstrated in experimental models, including cultures of rat aortic vascular smooth muscle cells [30] and bovine pulmonary artery smooth muscle cells [31]. The investigation revealed that these effects were attributable to the 5-HT2 and 5-HT4 receptors, respectively. Notably, the results of the in vitro investigation, particularly the culture “scratch” assay, are influenced not only by the cell migration rate, but also by the cell proliferation, which requires further studies to confirm the cell migration, rather than its division in the “culture wound.”

Conclusion

Activation of serotonin signaling mechanisms plays an important role in wound healing following various skin and subcutaneous tissue injuries by enhancing DF viability and proliferation, as well as viability, proliferation and migration of SAT-MSCs, as demonstrated in our study. Therefore, it can be hypothesized that serotonin or 5-HTR agonists may serve as promising candidates for enhancing cutaneous healing in injured patients. Importantly, the cellular response to serotonin signaling is tissue-specific and depends on the receptor subtype and intracellular signaling pathways mediating secondary messenger activation. The proliferative effects and activated migration of tumor cells and fibroblasts are mediated by the induction through 5-HTR1D and 5-HTR2B receptors, which are key mediators of the intracellular PI3K/AKT signaling, as well as the Wnt/β-catenin pathway. Together with mTOR-dependent inhibition of autophagy, these pathways promote positive regenerative effects and tumor progression. A more detailed elucidation of the mechanisms will facilitate the selective stimulation of skin wound regeneration without pro-oncogenic effects. Further experiments are required to investigate the function of serotonin and its receptor-mediated effects. This will contribute to a better understanding of the role of the serotonergic system in wound healing.

Additional Information

Author contributions: T.T. Chibirova: investigation, writing—original draft; R.I. Kokaev: project administration, writing—original draft, writing—review & editing; A.A. Islaev: investigation, visualization, writing—review & editing; G.S. Kokaev: investigation; S.V. Skupnevskii: project administration, writing—review & editing. All the authors approved the version of the manuscript to be published and agreed to be accountable for all aspects of the work, ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Acknowledgments: Not applicable.

Ethics approval: The study was approved by the Ethics Committee of the Institute of Biomedical Research, a branch of the Federal Research Center Vladikavkaz Scientific Centre of the Russian Academy of Sciences (Protocol No. 7, dated February 20, 2019). Biological material was collected in accordance with the guidelines and ethical standards of GOST R 53434-2009 Principles of Good Laboratory Practice.

Funding sources: No funding.

Disclosure of interests: The authors have no relationships, activities, or interests for the last three years related to for-profit or not-for-profit third parties whose interests may be affected by the content of the article.

Statement of originality: No previously published material (text, images, or data) was used in this work.

Data availability statement: All data generated during this study are available in the article.

Generative AI: No generative artificial intelligence technologies were used to prepare this article.

Provenance and peer review: This paper was submitted unsolicited and reviewed following the standard procedure. The review process involved two external reviewers, a member of the editorial board, and an in-house scientific editor.

About the authors

Tamara T. Chibirova

Vladikavkaz Scientific Centre of the Russian Academy of Sciences

Author for correspondence.
Email: tamaramerdenova@mail.ru
ORCID iD: 0000-0003-0819-8915
SPIN-code: 2736-0620
Russian Federation, RSO-Alania, Mikhailovskoye

Romesh I. Kokaev

Vladikavkaz Scientific Centre of the Russian Academy of Sciences

Email: romesh_k@mail.ru
ORCID iD: 0000-0002-2326-1348
SPIN-code: 5918-9041

MD, Cand. Sci. (Medicine), Associate Professor

Russian Federation, RSO-Alania, Mikhailovskoye

Altynbek A. Islaev

Vladikavkaz Scientific Centre of the Russian Academy of Sciences

Email: romesh_k@mail.ru
ORCID iD: 0000-0002-7800-8593
SPIN-code: 6435-1072
Russian Federation, RSO-Alania, Mikhailovskoye

Gavril S. Kokaev

Vladikavkaz Scientific Centre of the Russian Academy of Sciences

Email: zigavrik@gmail.com
ORCID iD: 0009-0001-8910-3537
SPIN-code: 3329-3030
Russian Federation, RSO-Alania, Mikhailovskoye

S. V. Skupnevskii

Vladikavkaz Scientific Centre of the Russian Academy of Sciences; North Ossetian State University

Email: dreammas@yandex.ru
ORCID iD: 0000-0002-6233-5944
SPIN-code: 7922-4399
Russian Federation, RSO-Alania, Mikhailovskoye; Vladikavkaz

References

Supplementary files