The Serotonin Transporter Promotes a Pathological Estrogen Metabolic Pathway in Pulmonary Hypertension via Cytochrome P450 1B1

Ethics

All experimental procedures were carried out in accordance with the United Kingdom Animal Procedures Act (1986) and with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication 85-23, revised 1996). Rodents were housed in a 12-hour light-dark cycle with access to food and water ad libitum. Experimental procedures using human PASMCs (hPASMCs) conform to the principles outlined in the Declaration of Helsinki. All animals were randomly allocated to groups, and all measurements and analysis were done in a blinded fashion, where possible.

Animal models and in vivo CYP1B1 inhibition with 2,3′,4,5′-tetramethoxystilbene

Female mice 5–6 months old ubiquitously overexpressing the human SERT gene (SERT⁺ mice) were generated as previously described on a C57Bl/6J CBA background. ³⁰ These were a kind gift from the late Professor Tony Harmar (University of Edinburgh). Age-matched littermates (hereafter referred to as wild-type) were studied as controls. Mice were subdivided into groups to receive either the highly potent and selective CYP1B1 inhibitor 2,3′,4,5′-tetramethoxystilbene (TMS; 1.5 mg/kg/day by intraperitoneal [i.p.] injection; Tocris Biosciences, Bristol, UK) or vehicle (~5% [v/v] ethanol in 0.9% saline) for 14 days before assessment of hemodynamic parameters.

Male and female Wistar rats (Harlan Laboratories, Bicester, UK), 10–12 weeks old at euthanasia received either the pneumotoxin MCT pyrrole (Sigma-Aldrich, Gillingham, UK) or its vehicle at a dose of 60 mg/kg by subcutaneous injection in the flank at day 0. MCT was dissolved in 1 mol/L hydrogen chloride (HCl) at a concentration of 100 mg/mL, neutralized with 1 mol/L sodium hydroxide (NaOH), and diluted with sterile distilled water to 6 mg/mL. All animals were weighed every day and monitored for any signs of discomfort. A single injection of MCT is sufficient to induce a PH phenotype in rats (MCT does not induce PH in mice). MCT mediates damage to the pulmonary endothelium and promotes the extension of smooth muscle cells into normally nonmuscular pulmonary arteries, promoting PH and right ventricular (RV) heart failure. ³¹ Fourteen days after MCT injection, animals were administered the CYP1B1 inhibitor TMS, at 3 mg/kg/day, or its vehicle (~5% [v/v] ethanol in 0.9% saline) by i.p. injection for an additional 14 days before hemodynamic analysis.

In vivo hemodynamic measurements

Rats and mice were anesthetized with isoflurane supplemented with a constant flow of oxygen. RV systolic pressures (RVSPs) were obtained by transdiaphragmatic puncture of the right ventricle with a saline-filled 25-gauge needle (in mice) and by jugular vein cannulation of the right ventricle with a fluid-filled catheter (in rats). To monitor off-target effects on the systemic vasculature, mean systemic arterial pressures (SAPs) were monitored by microcannulation of the left common carotid artery.

RV hypertrophy

RV hypertrophy was assessed by the Fulton index ³² by calculating the ratio of the dry weight of the right ventricle to the dry weight of the left ventricle plus septum.

Lung histopathology

The inferior lobe of the lung was fixed in 10% neutral buffered formalin for a minimum of 4 hours with gentle agitation before embedding in paraffin. Sections (3-μm) were cut and stained with elastin and counterstained with Picro Sirius Red for identification of vascular thickening in the small arteries with an internal diameter up to 80 μm. This was characterized by an increase in the vessel wall diameter in more than 50% of the arterial wall. The total number of remodeled vessels was expressed as a ratio of the total number of vessels present within a lung section.

CYP1B1 immunolocalization

CYP1B1 immunolocalization was determined in mouse, rat, and human pulmonary arteries by immunohistochemistry, as described previously. ⁵ Briefly, 3-μm sections were deparaffinized and rehydrated through a xylene-ethanol-to-water gradient. After antigen retrieval with citric acid buffer, endogenous peroxidase and biotin activity was blocked, followed by a 1-hour incubation with 2.5% normal horse serum to block nonspecific binding. Sections were incubated overnight at 4°C with an antibody against CYP1B1 (0.6 μg/mL, ab32649, Abcam, Cambridge, UK), proliferating cell nuclear antigen (PCNA; 0.2 μg/mL, ab2426, Abcam), SERT (1:200, whole antiserum, ab44520, Abcam) or α-smooth muscle cell actin (α-SMA; 0.4 μg/mL, ab5694, Abcam). Excess primary antibody was removed by 3 consecutive washes in Tris-buffered saline with Tween 20 (TBS-T) and then incubated with a secondary anti-rabbit antibody for 1 hour at room temperature. Cells positive for CYP1B1 were visualized by VIP peroxidase (which stains pink; Vector Laboratories, Peterborough, UK) in mice and diaminobenzidine (DAB, which stains dark brown; Vector Laboratories) in rats and humans. Cells positive for PCNA, SERT, and α-SMA were visualized by DAB. Rat sections were counterstained with hematoxylin. Images were captured with a Zeiss Axio Imager M1. For all antibodies, an immunoglobulin G control was performed at the same concentration to control for nonspecific binding.

PCNA quantification

The percentage of PCNA-expressing nuclei was calculated with a free web application (ImmunoRatio) used for automated analysis of immunostained tissue sections. The application segments DAB-stained and hematoxylin-stained nuclei regions from the user-submitted image and calculates the percentage of DAB-stained nuclear area in the total nuclear area. A total of 3 or 4 pulmonary arteries were analyzed per lung section.

Determination of reactive oxygen species in lung sections by immunofluorescence

Immunohistochemistry of the reactive oxygen species (ROS) marker 8-hydroxyguanosine (8-OHG) was determined in the lungs of wild-type, SERT⁺, and TMS-treated SERT⁺ mice. Sections were deparaffinized in a xylene-ethanol-to-water gradient. Antigen retrieval was performed by boiling slides in ethylenediaminetetraacetic acid (pH 8.0) for 15 minutes before a 1-hour incubation in a humidified chamber at room temperature with 10% donkey serum/1% bovine serum albumin in 1× TBS-T to block nonspecific binding. Sections were incubated with an antibody against 8-OHG overnight at 4°C (5 μg/mL, ab10802, Abcam). For identification of 8-OHG-positive cells, sections were incubated with Alexa-fluor-488-conjugated donkey anti-goat secondary antibody (A-11055, Molecular Probes, Life Technologies, Warrington, UK) for 45 minutes at room temperature in the dark. Lipofuscin-mediated autofluorescence was removed with 0.1% Sudan Black B (Sigma-Aldrich) in methanol w/v for 10 minutes. Slides were mounted with ProLong Gold antifade mounting media containing DAPI (4′,6-diamidino-2-phenylindole; P-36931, Molecular Probes, Life Technologies). Fluorescence imaging was measured in an Axiovert 200M microscope with a laser-scanning module LSM 510 (Carl Zeiss, Jena, Germany). DAPI was excited at 405 nm and Alexa-fluor 488 at 488 nm. Images were captured with the LSM 510 evaluation software physiology (Zeiss, Cambridge, UK). Mean fluorescence intensity of 8-OHG was measured on at least 5 different calibrated images of lung sections of the different groups at ×40 magnification with ImageJ software. An auto threshold for each image was applied by using the Huang method in Image J.

Protein analysis

Whole-lung samples were homogenized and lysed in ice-cold 0.1% lauryl maltoside solution in phosphate-buffered saline (PBS; v/v), and protein concentrations were measured by a bicinchoninic acid (BCA) assay (Thermo Scientific, Cramlington, UK). Protein (30 μg) was loaded for whole-lung homogenates, and samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Protein expression of CYP1B1 (2 μg/mL; sc-32882, Santa Cruz Biotechnology, Dallas) and SERT expression (2 μg/mL; AB1594P, Millipore, Livingston, UK) was determined by overnight incubation at 4°C and normalized to GAPDH (0.2 μg/mL; ab8264, Abcam).

Estrogen immunoassay

The levels of estrogen were determined by enzyme-linked immunosorbent assay (ELISA) in whole-lung homogenate samples from female wild-type and SERT⁺ mice and SERT⁺ mice treated with TMS. Lung samples were homogenized in ice-cold 1% lauryl maltoside solution in PBS (v/v) and solubilized on ice; cellular fractionation followed. Protein concentrations were determined by a BCA assay. Protein (400 μg) was loaded for mouse lung samples and assayed in duplicate as per manufacturer's instructions (Cayman Chemical, Ann Arbor, MI). The plate was read at a wavelength of 405 nm for end point measurements (SpectraMax M2 plate reader, Molecular Devices, Sunnyvale, CA). Data were normalized to per microgram of protein.

Human PASMCs

Human PASMCs (hPASMCs) were provided by Professor Nicholas W. Morrell (University of Cambridge). Female hPASMCs were explanted from the distal pulmonary microvasculature from subjects with no reported presence of PAH. Three independent female cell lines were used in this study and characterized by morphological assessment and staining for the smooth muscle cell marker α-SMA. Assays were performed between passages 2 and 7. Cells were seeded in 24-well plates (cell counts) and 96-well plates (BrdU [bromodeoxyuridine] incorporation) at a density of 7,500 cells/cm². Cells were grown to ~60% confluency and then cell-cycle synchronized by serum deprivation (0.2% charcoal-stripped fetal bovine serum [FBS]) in phenol red–free Dulbecco's modified Eagle medium (DMEM; Invitrogen, Loughborough, UK) for 24 hours.

Charcoal-stripped FBS

FBS (Sera Labs, Haywards Heath, UK) was charcoal stripped twice to remove estrogens. Dextran-coated charcoal (Sigma-Aldrich) was added to FBS at a concentration of 1 g/100 mL and left overnight at 4°C under gentle agitation. Samples were centrifuged at 1,811g at 4°C for 30 minutes. The stripped serum was decanted and filtered through a 0.22-μm filter. ELISA analysis of 50 μL of FBS confirmed successful removal of estrogen (as described above).

Cell proliferation assays

Proliferation of hPASMCs was assessed by two approaches: live cell counts (cell viability) with a hemocytometer and a BrdU incorporation assay, as per manufacturers protocol (Millipore). Cells were treated for 72 hours, in phenol red–free 2% charcoal-stripped FBS DMEM with 1 nM 16α-OHE1 (Steraloids, Newport, RI) and 0.1 μM MPP dihydrochloride (estrogen receptor alpha [ERα] antagonist; Tocris) or 0.002 pg/mL ERα antibody (sc-7207, Santa Cruz Biotechnology). Antagonists/antibodies were added 30 minutes before the addition of 16α-OHE1. Cell culture media and drugs were replaced after 48 hours, and PASMC proliferation was assessed at 72 hours.

Statistics

Data are represented as the group mean ± SEM. Data were analyzed by either a Student 2-tailed t test or a 1-way analysis of variance followed by a Tukey post hoc test. The statistical test used is indicated in the figure legends.

Female SERT⁺ mice develop PH via CYP1B1

Female mice overexpressing the human SERT gene develop a spontaneous PH phenotype that is dependent on estrogen. ¹³ As serotonin can modulate CYP1B1 expression ²⁸ and CYP1B1 contributes to the development of PH, ⁵ we investigated whether the development of PH in these mice was related to increased CYP1B1 activity. Overexpression of SERT resulted in elevated RVSPs (reflective of PH) compared to those in wild-type mice, and CYP1B1 inhibition with the highly potent and selective inhibitor TMS attenuated this effect (Fig. 1A). As previously reported, ¹³ female SERT⁺ mice did not develop RV hypertrophy, despite elevated RVSPs. This is consistent with observations in numerous transgenic models of PH in normoxic conditions,^{12,18,33–35} and no further effects were observed in mice treated with TMS (Fig. 1B). Overexpression of SERT and CYP1B1 inhibition had no effect on mean SAPs (Fig. S1; Figs. S1 and S2 are available online). SERT⁺ mice had lower heart rates than wild-type mice, and this was normalized by CYP1B1 inhibition (Fig. 1C). SERT⁺ mice had a significant degree of pulmonary vascular remodeling within the distal vasculature (where arteries are normally composed of a thin vascular layer), and this was attenuated by CYP1B1 inhibition (Fig. 1D).

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Figure 1.

Inhibition of cytochrome P450 1B1 (CYP1B1) with 2,3′, 4,5′-tetramethoxystilbene (TMS) attenuates PH in female SERT⁺ mice. Female SERT⁺ mice were injected with 1.5 mg/kg TMS by intraperitoneal injection for 2 weeks and compared with wild-type (WT; C57Bl/6J CBA) and SERT⁺ vehicle-treated mice for its effects on PH indices. A, Right ventricular systolic pressure (RVSP) measurement per group, as measured by transdiaphragmatic puncture; n = 9–10. B, Right ventricular hypertrophy expressed as the ratio of the weight of the right ventricle (RV) to the combined weight of the left ventricle and septum (LV+S); n = 10–11. C, Heart rate; n = 7–9. D, Pulmonary vascular remodeling; n = 5–6. E, Representative images of pulmonary vascular remodeling in arteries for each group. Scale bar: 20 μm. Data were analyzed by a 1-way analysis of variance followed by a Tukey post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001. SERT⁺: serotonin transporter is overexpressed.

CYP1B1 expression was increased in SERT⁺ mice, and CYP1B1 inhibition increased estrogen levels in the lung

Overexpression of the human SERT gene in mice increased CYP1B1 protein expression in whole-lung homogenates compared with that in wild-type mice. CYP1B1 expression in SERT⁺ TMS-treated mice was not significantly different from that in wild-type mice, suggesting that TMS reduced CYP1B1 expression. These findings were confirmed by immunohistochemistry staining for CYP1B1 (Fig. 2A, 2B).

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Figure 2.

Cytochrome P450 1B1 (CYP1B1) expression is increased in female SERT⁺ mice, and CYP1B1 inhibition increases estrogen concentrations in lung homogenates. Female SERT⁺ mice were injected with 1.5 mg/kg 2,3′,4,5′-tetramethoxystilbene (TMS) by intraperitoneal injection for 2 weeks and compared with wild-type (WT; C57Bl/6J CBA) and SERT⁺ vehicle-treated mice. A, Whole-lung homogenates (30 μg) were analyzed for CYP1B1 expression by Western blotting and normalized to GAPDH; n = 5–8. B, Representative CYP1B1 immunolocalization in pulmonary arteries, as determined by VIP peroxidase–positive cells (pink). Scale bar: 20 μm. C, Estrogen (17β-estradiol) concentrations as determined by enzyme-linked immunosorbent assay in 400 μg of whole-lung homogenates; n = 4–5. Data were analyzed by a 1-way analysis of variance followed by a Tukey post hoc test. ***P < 0.001. SERT⁺: serotonin transporter is overexpressed.

A recent estrogen metabolic analysis of Cyp1b1-knockout mice reported increased lung concentrations of estrogen in Cyp1b1-knockout mice compared with wild-type mice. ³⁶ CYP enzymes regulate estrogen metabolism and it is therefore plausible that the increase in estrogen concentrations in these mice is attributed to reduced metabolism by CYP1B1. It was therefore of interest to assess estrogen concentrations in the lungs of SERT⁺ mice to determine the effect of SERT overexpression and CYP1B1 inhibition. Whole-lung estrogen concentrations were determined by ELISA and found to be similar in SERT⁺ mice and wild-type mice. However, CYP1B1 inhibition in SERT⁺ mice increased estrogen concentrations (Fig. 2C).

Oxidative stress is increased in SERT⁺ lungs

The formation of ROS by the metabolism of estrogen by CYP1B1 has been previously described, ³⁷ and increased oxidative stress may be associated with the pathobiology of PAH. ³⁸ We therefore investigated whether oxidative stress was regulated by CYP1B1 activity in our model by immunohistochemical analysis of 8-OHG (a sensitive measure of ROS). Female SERT⁺ mice had increased levels of 8-OHG compared to wild-type mice, and CYP1B1 inhibition with TMS had no effect on 8-OHG levels (Fig. 3). There was expression of 8-OHG within numerous cellular compartments in SERT⁺ mice treated with vehicle and TMS, including alveoli, bronchi, bronchioles, and vessels, whereas expression was visually less in control mice (Fig. 3). And 8-OHG was observed in both the cytoplasm and the nucleus, indicating increased RNA and DNA oxidation.

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Figure 3.

SERT⁺ mice have increased RNA and DNA oxidative damage in the lungs. A, Lung sections obtained from wild-type (WT) and SERT⁺ mice and SERT⁺ mice treated with the cytochrome P450 1B1 (CYP1B1) inhibitor 2,3′,4,5′-tetramethoxystilbene (TMS) were immunostained for the oxidative-stress marker 8-hydroxyguanosine (8-OHG). Sections were counterstained with the nuclear stain DAPI (4′,6-diamidino-2-phenylindole). Scale bar: 50 μm. B, 8-OHG mean fluorescence intensity was measured in whole-lung sections with ImageJ software; n = 4–5. Data were analyzed by a 1-way analysis of variance followed by a Tukey post hoc test. *P < 0.05; **P < 0.01. SERT⁺: serotonin transporter is overexpressed.

CYP1B1 inhibition did not improve RV function in MCT-induced PH but prolonged survival

Previous studies have reported increased SERT expression in the MCT model of PH. ²⁹ Because the overexpression of human SERT increased CYP1B1 expression in our mouse model, we rationalized that CYP1B1 expression would also be increased by MCT. Whole-lung expression of SERT protein was increased in female but not in male rats (Fig. 4A). However, CYP1B1 protein expression (Fig. 4B) was unchanged in the lungs from both male and female rats that had been treated with MCT for 28 days (all tissue examined was taken from animals that survived until day 28). MCT induced significant increases in RVSP (Fig. 5A) and RV hypertrophy (Fig. 5B). TMS had no effects on RVSP (Fig. 5A) and only moderate, yet significant, effects in reducing RV hypertrophy in male rats, while no effects were observed in female rats (Fig. 5B). Male rats developed more severe RV hypertrophy than female rats in response to MCT, despite developing a similar degree of increased RVSP (Fig. 5A, 5B). MCT and TMS treatment had no effects on mean SAPs in female and male rats (Fig. S2). MCT lowered the heart rate in male rats, and this was normalized by TMS to pressures similar to those in vehicle-treated rats. No changes in heart rate were observed in female rats (Fig. 5C). Mortalities were reported 23 and 26 days after MCT administration in male and female rats, respectively. TMS prolonged survival in male rats, and no mortalities were reported in female rats treated with TMS 2 weeks after administration of MCT (Fig. 5D). MCT induced a significant degree of pulmonary vascular remodeling in the distal vasculature, and this was not affected by CYP1B1 inhibition (Fig. 6A, 6B). CYP1B1 immunopositive cells were predominantly observed in pulmonary arterial endothelial cells (Fig. 6C).

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Figure 4.

Serotonin transporter (SERT) and cytochrome P450 1B1 (CYP1B1) expression in monocrotaline (MCT)-treated rats. Female and male rats were given a single dose of monocrotaline (MCT; 60 mg/kg subcutaneously), and 28 days later whole-lung homogenates (30 μg) were analyzed for protein expression by Western blotting for SERT, n = 6 (A), and CYP1B1, n = 8 (B). Data were normalized to GAPDH. Data were analyzed by an unpaired t test. *P < 0.05. Veh: vehicle.

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Figure 5.

Inhibition of cytochrome P450 1B1 (CYP1B1) with 2,3′, 4,5′-tetramethoxystilbene (TMS) does not reverse monocrotaline (MCT)-induced PH yet improves survival. Female and male rats were given a single dose of MCT (60 mg/kg subcutaneously) and 14 days later were given TMS (3 mg/kg/day intraperitoneally) or vehicle (~5% ethanol in saline) for an additional 14 days and then assessed for indices of PH. A, Right ventricular systolic pressures (RVSP) as assessed by jugular vein cannulation; n = 5–11. B, Right ventricular hypertrophy as assessed by ratio of the weight of the right ventricle (RV) to the combined weight of the left ventricle and septum (LV+S); n = 9–14. C, Heart rate; n = 6–12. D, Kaplan-Meier survival analysis in rats treated with MCT (total of 16 animals per group at the start of the experiment). Data were analyzed by a 1-way analysis of variance followed by a Tukey post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001, vehicle versus MCT; ^#P < 0.05, male versus female.

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Figure 6.

Inhibition of cytochrome P450 1B1 (CYP1B1) with 2,3′, 4,5′-tetramethoxystilbene (TMS) does not reverse monocrotaline (MCT)-induced pulmonary arterial remodeling. Female and male rats were given a single dose of MCT (60 mg/kg subcutaneously) and 14 days later were given TMS (3 mg/kg/day intraperitoneally) or vehicle (~5% ethanol in saline) for an additional 14 days and then assessed for indices of PH. A, Pulmonary vascular remodeling; n = 6–7. Data were analyzed by a 1-way analysis of variance followed by a Tukey post hoc test. **P < 0.01; ***P < 0.001. B, Representative pulmonary arteries for each group. Scale bar: 20 μm. C, CYP1B1 immunolocalization in representative pulmonary arteries indicates positive cells in the endothelial cell layer and periad-ventitial layer, as visualized by diaminobenzidine (dark brown). Sections were counterstained with hematoxylin. Scale bar: 50 μm.

The CYP1B1 metabolite 16α-OHE1 stimulates PASMC proliferation via ERα

PASMC proliferation is an established and dominant feature associated with PAH pathobiology,^26,27 and we have previously shown that estrogen induces hPASMC proliferation via CYP1B1. ⁵ Using immunohistochemistry, we determined that both SERT and CYP1B1 are highly expressed in α-SMA-positive cells (PASMCs) in pulmonary arteries from patients with PAH, whereas minimal expression is observed in non-PAH arteries (Fig. 7). SERT overexpression in mice increased the expression of CYP1B1, suggesting that the PH phenotype observed in these mice may be mediated by increased cellular proliferation. To investigate this, we performed immunohistochemistry analysis of the DNA replication marker PCNA. PCNA expression was increased in SERT⁺ mice, and this was attenuated by CYP1B1 inhibition (Fig. 8A, 8B). We next investigated whether the proliferative CYP1B1 estrogen metabolite 16α-OHE1 promoted proliferation via the estrogen receptor ERα by selectively blocking ERα with a specific antagonist and antibody. The 16α-OHE1 induced significant increases in PASMC proliferation, and this was completely attenuated by blocking of ERα signaling by both a specific antagonist and an antibody, as assessed by cell counts (Fig. 8C, 8E) and BrdU incorporation (Fig. 8D, 8F). To prove that these effects were directly related to inhibition of 16α-OHE1 signaling through ERα and not by indirect effects mediated by the antagonist or antibody, we stimulated hPASMCs with each treatment alone and observed no effects (data not shown).

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Figure 7.

Serotonin transporter (SERT) and cytochrome P450 1B1 (CYP1B1) are expressed in α-smooth muscle actin (α-SMA)-positive cells in human pulmonary arteries. Immunolocalization of SERT, CYP1B1 and α-SMA in human pulmonary arteries of non-PAH and PAH lung sections as visualized by diaminobenzidine (dark brown). PAH lung samples were from a female aged 26 years with heritable PAH (top) and a female aged 51 years with idiopathic PAH (bottom). Scale bar: 200 μm. PAH: pulmonary arterial hypertension.

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Figure 8.

The cytochrome P450 1B1 (CYP1B1) metabolite 16α-hydroxyestrone (16α-OHE1) induces cell proliferation via estrogen receptor α (ERα). A, Representative images of immunohistochemistry analysis of the DNA replication marker PCNA in wild-type (WT) mice and in SERT⁺ mice treated with vehicle or the CYP1B1 inhibitor 2,3′,4,5′-tetramethoxystilbene (TMS). Scale bar: 20 μm. B, Percentage PCNA-positive nuclei; n = 3–6, 3 or 4 pulmonary arteries analyzed per lung. Data were analyzed by a 1-way analysis of variance (ANOVA) followed by a Tukey post hoc test. C, D, Female human pulmonary arterial smooth muscle cells (PASMCs) were incubated with 16α-OHE1 for 72 hours in 2% charcoal-stripped serum in the presence or absence of ERα inhibition and assessed for proliferation by cell counts and BrdU incorporation. 16α-OHE1 induced PASMC proliferation in the presence of a selective ERα antagonist MPP dihydrochloride (0.1 μM), as assessed by cell counts, n = 15 replicates per group (C), and BrdU incorporation, n = 11 replicates per group (D). E, F, 16α-OHE1 induced PASMC proliferation in the presence of a specific ERα antibody (0.002 pg/mL) as assessed by cell counts, n = 6–9 replicates per group (E), and BrdU incorporation, n = 16 replicates per group (F). Data were analyzed by a 1-way ANOVA followed by a Tukey post hoc test. **P < 0.01; ***P < 0.001. BrdU: bromodeoxyuridine; FBS: fetal bovine serum; PCNA: proliferating cell nuclear antigen; SERT⁺: serotonin transporter is overexpressed.