FTO/m6A Regulate Prenatal Nicotine-Induced eNOS Uncoupling and BP Susceptibility

Abstract

BACKGROUD:

Prenatal nicotine exposure from maternal smoking or e-cigarette use increases cardiovascular risk in offspring, but the underlying mechanisms remain unclear. We tested whether fat mass and obesity-associated protein (FTO)-mediated N6-methyladenosine (m6A) RNA demethylation drives endothelial nitric oxide synthase (eNOS) uncoupling and programmed hypertensive susceptibility following prenatal nicotine exposure.

METHODS:

Using a clinically relevant rat model of chronic intermittent nicotine aerosol (CINA) exposure or saline aerosol control during gestational days 4 to 21, experiments were performed in adult offspring and human umbilical vein endothelial cells (HUVECs).

RESULTS:

Prenatal CINA programmed a male-specific increase in hypertensive susceptibility without altering baseline blood pressure. Adult male offspring exhibited enhanced pressor responses to vasoconstrictor stimuli, impaired endothelium-dependent vasodilation, paradoxical eNOS overexpression, reduced nitric oxide (NO) bioavailability, increased superoxide and hydrogen peroxide production, impaired eNOS dimerization, and decreased tetrahydrobiopterin (BH4) levels associated with reduced GTP cyclohydrolase-1(GTPCH1) expression, consistent with eNOS uncoupling. Mechanistically, prenatal CINA increased vascular FTO expression and induced site-specific m6A hypomethylation of eNOS mRNA. Pharmacological inhibition of FTO with FB23-2 restored eNOS and GTPCH1 expression, improved eNOS dimerization, and normalized NO production in mesenteric arteries. In HUVECs, nicotine exposure recapitulated FTO-dependent eNOS uncoupling, which was reversed by FB23-2 or siRNA-mediated FTO knockdown.

CONCLUSIONS:

Prenatal nicotine exposure disrupts endothelial redox homeostasis through FTO-dependent m6A RNA demethylation, leading to BH4 deficiency, eNOS uncoupling, and vascular dysfunction in male offspring. The FTO–BH4–eNOS pathway represents a novel epitranscriptomic mechanism underlying programmed hypertensive susceptibility and a potential therapeutic target for offspring exposed to nicotine during development.

Graphical Abstract

Introduction

Cardiovascular diseases (CVDs) remain the leading cause of mortality worldwide, emphasizing the need to identify early-life determinants of vascular health. The Developmental Origins of Health and Disease (DOHaD) framework posits that adverse prenatal exposures program long-term susceptibility to chronic conditions, including hypertension.¹ Maternal cigarette smoking is a well-established prenatal risk factor,² and the rapid expansion of non-combustible nicotine products, such as e-cigarettes and heat-not-burn devices, has intensified concerns regarding prenatal nicotine exposure.^3–5 Epidemiological and preclinical evidence indicates that gestational nicotine exposure increases offspring risk of hypertension and cardiovascular dysfunction in adulthood.^6,7 As a readily placentally transferable teratogen, nicotine is a key mediator of developmental cardiovascular reprogramming.^3,4,8 However, the sex-specific vascular consequences of chronic intermittent nicotine aerosol (CINA) exposure, a pattern that closely mirrors cigarette smoking and vaping,⁹ remain poorly defined.

Endothelial dysfunction is a critical early event in CVD pathogenesis and is characterized by reduced nitric oxide (NO) bioavailability and increased oxidative stress.¹⁰ Endothelial nitric oxide synthase (eNOS) is the principal source of vascular NO and regulates vascular tone and pressor responses to vasoactive stimuli.¹¹ Under physiological conditions, dimeric eNOS produces NO in a tetrahydrobiopterin (BH4)-dependent manner.¹² BH4 deficiency leads to eNOS uncoupling, whereby the enzyme shifts from NO generation to the production of reactive oxygen species (ROS), including superoxide (O₂^˙−) and hydrogen peroxide (H₂O₂).^12–15 Importantly, eNOS uncoupling can occur despite preserved or increased eNOS expression when BH4 availability is insufficient.^16–18 Reduced expression or activity of GTP cyclohydrolase I (GTPCH1), the rate-limiting enzyme in BH4 biosynthesis, is a major contributor to BH4 deficiency and may be exacerbated by oxidative stress from sources such as NADPH oxidase 2 (NOX2).^19–21 Smoking has been shown to reduce BH4 bioavailability and promote eNOS uncoupling in humans,²² yet whether prenatal CINA exposure induces persistent, sex-specific eNOS uncoupling in offspring resistance arteries remains unknown.

The persistence of developmental programming effects suggests involvement of stable epigenetic mechanisms.²³ In addition to DNA methylation and histone modifications, epitranscriptomic regulation via RNA modifications provides a dynamic layer of post-transcriptional control responsive to environmental stimuli.²⁴ N6-methyladenosine (m6A) is the most abundant internal mRNA modification and regulates mRNA stability, translation, and decay.^25–28 m6A dynamics are controlled by writer, reader, and eraser proteins, including the demethylase fat mass and obesity-associated protein (FTO).^29–32 FTO has been implicated in adult cardiovascular diseases such as heart failure, hypertension, and atherosclerosis;^33–36 however, its role in developmental nicotine programming and sex-specific endothelial dysfunction has not been examined. Whether FTO-mediated m6A demethylation drives post-transcriptional dysregulation of eNOS and GTPCH1 during prenatal nicotine exposure remains unknown.

We therefore hypothesized that prenatal CINA exposure programs endothelial dysfunction and hypertension via an FTO/m6A epitranscriptomic mechanism. We postulated that increased FTO activity disrupts post-transcriptional regulation of eNOS and GTPCH1, leading to BH4 depletion, eNOS uncoupling, and a pathological shift in vascular redox balance. Using a clinically relevant prenatal CINA model combined with molecular and functional rescue approaches, we identify a novel sex-dimorphic pathway linking developmental nicotine exposure to adult hypertension and highlight the FTO/m6A–derived BH4/eNOS coupling axis as a potential therapeutic target.

Methods

The data that support the findings of this study are available from the corresponding author upon reasonable request. All data are included in the article and its Supplemental Material, which also provides detailed experimental methods and procedures.

Pregnant rat model of CINA exposure

All experimental animal procedures were conducted in accordance with the National Institutes of Health (US) Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committees of Loma Linda University and the University of California, Los Angeles. Time-dated pregnant Sprague–Dawley rats were obtained from Charles River Laboratories (Portage, MI) and housed under controlled temperature conditions with a 12 h light/dark cycle, with ad libitum access to standard chow and water.

The CINA exposure protocol was adapted from our previous studies.^9,37 Briefly, a total of 37 timed-pregnant Sprague–Dawley rats were used and randomly assigned on gestational day 4 to either the nicotine aerosol exposure group (n=19 dams) or the control group (n=18 dams). Exposure occurred during the 12-hour dark cycle from GD4 until term (GD21). Nicotine aerosol was generated using a nebulizer containing a 0.2% nicotine solution. Nicotine aerosol delivery was calibrated to achieve maternal plasma nicotine and cotinine concentrations comparable to those observed in human smokers (nicotine: 10–35 ng/mL; cotinine: 100–240 ng/mL).³⁷ Experiments were performed in both male and female adult offspring at approximately 6 months of age.

For in vivo hemodynamic studies, offspring were anesthetized with infusion of 5% isoflurane (induction) and maintained with 2% isoflurane, and arterial blood pressure and heart rate were continuously recorded using a PowerLab 16/SP data acquisition system with Chart software (AD Instruments). For ex vivo and molecular studies, animals were euthanized under deep anesthesia with infusion of 5% isoflurane, followed by rapid tissue collection for vascular functional, biochemical, and molecular analysis.

Statistical analysis

Data are presented as mean ± SEM. Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA). Data normality was assessed using the Shapiro–Wilk test (n≥3), and variance homogeneity was evaluated using the F test (two groups) or the Brown–Forsythe test (multiple groups). For two-group comparisons, unpaired two-tailed Student’s t-test was used when data were normally distributed with equal variances; otherwise, the Mann–Whitney U test was applied. For multiple-group comparisons, one-way, two-way, or two-way repeated-measures ANOVA was performed as appropriate, followed by Bonferroni post hoc tests. Greenhouse–Greenhouse–Geisser correction was applied for repeated-measures factors with more than two levels. Pearson’s correlation coefficient was used for correlation analyses. A two-tailed P value <0.05 was considered statistically significant. The exact statistical tests and corresponding sample sizes for each experiment are reported in the respective figure legends.

Result

1. Prenatal CINA exposure induces sex-dependent enhancement of vasoconstrictor-induced blood pressure responses and attenuation of vasodilator responses in adult offspring

To investigate the long-term effects of prenatal chronic inhalation of nicotine aerosol (CINA) exposure on arterial pressure regulation, baseline and acute blood pressure (BP) responses to vasoactive agents were assessed in anesthetized adult (6-month-old) offspring. Baseline systolic (SBP), diastolic (DBP), and mean arterial pressure (MAP), were not affected by prenatal CINA exposure in either male or female offspring (Figure S1A and B). Acute intravenous bolus administration of angiotensin II (Ang II) produced dose-dependent increases in MAP in all groups (Figure 1A and B). Notably, prenatal CINA exposure significantly augmented Ang II-induced increases in MAP (ΔMAP) in male offspring compared with saline-exposed male controls at both 50 and 100 ng/kg doses (Figure 1A). In contrast, Ang II-induced ΔMAP were comparable between CINA-exposed and saline-treated female offspring (Figure 1B). Similarly, acute phenylephrine (PE, 20 μg/kg) administration elicited a significantly greater ΔMAP in male CINA offspring than in male controls, whereas no significant differences were observed in females (Figure S1C).

Figure 1. Prenatal CINA exposure induces sex-specific alterations in blood pressure responses in adult offspring.

Mean arterial pressure (MAP) responses to serial intravenous bolus injections of angiotensin II (Ang II; 10–100 ng/kg) and acetylcholine (Ach; 1–10 μg/kg) were assessed in adult offspring prenatally exposed to saline aerosol or chronic intermittent nicotine aerosol (CINA). A and B, Ang II-induced dose-dependent changes in MAP (ΔMAP) in male (A; n=7/group) and female (B; n=6/group) offspring. C and D, Ach-induced dose-dependent changes in MAP in male (C; n=8/group) and female (D; Saline, n=5; CINA, n=6) offspring. E, Effect of NOS inhibition with L-NAME (20 mg/kg) on Ach-induced ΔMAP in male offspring (n=6/group). F, Ang II (100 ng/kg)-induced ΔMAP in male offspring in the absence and presence of L-NAME (20 mg/kg) (n=5/group). Data are presented as mean ± SEM. For dose-response experiments (A-E), two-way repeated-measures ANOVA was performed separately for each sex, with prenatal treatment as the between-subjects factor and dose as the within-subjects factor, followed by Bonferroni multiple-comparison test. Panel F was analyzed by two-way repeated-measures ANOVA with prenatal treatment as the between-subject factor and L-NAME condition (absent vs present) as the within-subject factor, followed by Bonferroni multiple-comparison test. Normality and homogeneity of variance were assessed using the Shapiro-Wilk and Brown-Forsythe tests, respectively. The Greenhouse-Geisser correction was applied to panels A-E. n values indicate the number of offspring per group. ns, not significant.

Endothelium-dependent BP responses were next evaluated using acetylcholine (Ach), with and without nitric oxide synthase (NOS) inhibition by Nω-nitro-L-arginine methyl ester (L-NAME). Prenatal CINA exposure caused dose-dependent attenuation of Ach-induced reductions in MAP in male (Figure 1C), but not in female (Figure 1D) offspring, compared with their respective saline controls. Importantly, the differences in Ach-induced ΔMAP responses between saline and CINA groups in males were abolished following acute L-NAME administration (20 mg/kg) (Figure 1E). Consistent with impaired NO signaling, Ang II (100 ng/kg)–induced ΔMAP responses remained significantly higher in male CINA offspring than in controls (Figure 1F). L-NAME pretreatment markedly enhanced Ang II–induced ΔMAP responses in saline control males; however, this potentiating effect was significantly attenuated in prenatal CINA-exposed males (Figure 1F). Collectively, these findings indicate that prenatal CINA exposure programs sex-specific alterations in BP regulation, at least in part through disruption of endothelial NO signaling.

2. Prenatal CINA exposure sex-dependently impairs Ach-induced vasorelaxation and is associated with paradoxical eNOS overexpression in male offspring

Based on in vivo evidence of sex-specific alterations in NO-mediated BP regulation, we hypothesized that prenatal CINA exposure impairs endothelium-dependent vasorelaxation in resistance vessels, preferentially in males. Using pressure myography, freshly isolated mesenteric resistance arteries were examined. In male offspring, prenatal CINA exposure significantly attenuated Ach-induced vasorelaxation compared with saline controls (Figure 2A). This difference was abolished by L-NAME treatment, indicating dependence on NOS activity (Figure 2C). In contrast, prenatal CINA exposure did not affect Ach-induced relaxation in female offspring, either in the absence (Figure 2B) or presence (Figure 2D) of L-NAME. Endothelium-independent vasodilation to sodium nitroprusside (SNP) was unchanged by prenatal CINA exposure in males (Figure 2E), confirming preserved smooth muscle responsiveness to NO.

Figure 2. Prenatal CINA exposure impairs endothelium-dependent vasodilation and increases eNOS protein expression selectively in male offspring.

Endothelium-dependent relaxation of mesenteric artery (MA) rings in response to acetylcholine (Ach) was assessed in adult offspring. A and B, Ach-induced relaxation in male (A; Saline, n=9; CINA, n=13) and female (B; Saline, n=7; CINA, n=11) offspring. C and D, Ach-induced relaxation in male (C; Saline, n=8; CINA, n=9) and female (D; Saline, n=4; CINA, n=6) offspring in the presence of L-NAME (100 μM). E, Endothelium-independent relaxation to sodium nitroprusside (SNP) in male offspring (Saline, n=4; CINA, n=6). F, Representative immunoblots and quantitative analysis of eNOS protein expression in MAs from male and female offspring (n=5 offspring per sex per group). GAPDH or β-actin served as loading controls. Each vessel ring was obtained from a single offspring, and n values indicate the number of offspring per group. The same animals were used across all concentrations within each experiment. Data are presented as mean ± SEM. For panels A–E, two-way repeated-measures ANOVA was performed with ACh or SNP concentration as the within-subjects factor and prenatal treatment as the between-subjects factor, followed by Bonferroni multiple-comparison test. The Greenhouse–Geisser correction was applied to panels A–E. Panel F was analyzed using an unpaired two-tailed Student’s t test. Normality was assessed using the Shapiro–Wilk test, and homogeneity of variance was assessed using the Brown–Forsythe test (A-E) or the F test (F). *P<0.05, **P<0.01 versus saline aerosol; ns, not significant.

Given the central role of eNOS in NO production,¹² we examined eNOS protein expression. Surprisingly, Western blot analysis revealed a marked (~2.6-fold) increase in total eNOS protein abundance in mesenteric arteries from male CINA offspring compared with controls (Figure 2F). No changes in eNOS expression were observed in females (Figure 2F). This paradoxical upregulation of eNOS in the presence of impaired NO-mediated vasodilation suggests sex-specific disruption of eNOS coupling in male offspring.

3. Prenatal CINA exposure promotes vascular eNOS uncoupling, resulting in NO deficiency and ROS overproduction in a sex-dependent manner.

To determine whether prenatal CINA exposure induces eNOS uncoupling, we assessed NO bioavailability and ROS production in mesenteric arteries (MAs). Nitrate/nitrite levels, a surrogate marker of NO production, were significantly reduced in MAs from male CINA offspring compared with male controls (Figure 3A, left panel), whereas no differences were observed in females (Figure 3A, right panel). In contrast, total ROS production was significantly elevated in both MAs (Figure 3B) and aortas (Figure 3C) from male CINA offspring. Dihydroethidium (DHE) fluorescence microtopography demonstrated approximately 1.6-fold higher endothelial DHE-detectable O₂^˙−-dependent signal in MAs from male CINA offspring (Figure 3D). L-NAME significantly reduced endothelial DHE-detectable O₂^˙− -dependent signal levels in CINA-exposed males, but not in controls, abolishing group differences (Figure 3D), implicating uncoupled eNOS as a major ROS source.

Figure 3. Prenatal CINA exposure promotes eNOS uncoupling, nitric oxide deficiency, and ROS overproduction in male offspring.

A, Nitrate/nitrite (NOx) levels in MAs from male (left) and female (right) offspring (n=6 offspring per sex per group) and were normalized to total tissue protein content and expressed as fold change relative to the saline-treated control group. B and C, Total ROS production in MAs (B) and aorta (C) from male offspring (n=5 offspring per group). D, Endothelial superoxide-dependent DHE fluorescence in third-order MAs from male offspring in the absence or presence of L-NAME (500 μM) (n=7 offspring per group). E, H₂O₂ production in MAs from male (left) and female (right) offspring measured by Amplex Red assay in the absence or presence of L-NAME (100 μM) (n=5 offspring per group). F, Ach-induced relaxation in male MAs in the presence of PEG-catalase (400 U/mL). Vessels were preconstricted with U46619 (200 nM). Saline, n=4 offspring; CINA, n=9 offspring. One vessel ring was obtained from each offspring, and the same animals were used across all concentrations. Data are presented as mean ± SEM. Normality was assessed using the Shapiro-Wilk test. Homogeneity of variance was assessed using the F test for panels A-C and the Brown-Forsythe test for panels D-F. Group comparisons were performed as follows: panels A-C, unpaired two-tailed Student’s t test; panels D and E, two-way ANOVA to assess the effects of prenatal exposure and L-NAME treatment, with panel E analyzed separately by sex, followed by Bonferroni multiple-comparison test; panel F, two-way repeated-measures ANOVA (Ach concentration: within-subjects; prenatal exposure: between-subjects) with the Greenhouse–Geisser correction followed by Bonferroni multiple-comparison test.. ns, not significant.

Because eNOS uncoupling is associated with increased H₂O₂ production,^38–41 H₂O₂ levels were quantified using Amplex Red. Male CINA offspring exhibited significantly elevated basal H₂O₂ production in MAs compared with controls (Figure 3E, left panel). L-NAME treatment reduced H₂O₂ levels in both groups, eliminating the difference (Figure 3E, left panel). In contrast, basal H₂O₂ levels were similar in female offspring regardless of treatment (Figure 3E, right panel). Functionally, pretreatment with the H₂O₂ scavenger catalase–polyethylene glycol (PEG-catalase) abolished the difference in Ach-induced vasodilation between saline and CINA-exposed male offspring (Figure 3F). These findings demonstrate that prenatal CINA exposure induces sex-specific eNOS uncoupling, resulting in increased ROS, particularly H₂O₂, which contributes to endothelial dysfunction in males.

4. Prenatal CINA exposure induces sex-specific downregulation of GTPCH1, BH4 deficiency, and impaired eNOS dimerization in male offspring mesenteric arteries

Because eNOS uncoupling is characterized by reduced eNOS dimerization,^12,16 eNOS dimer/monomer ratios were assessed using low-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Prenatal CINA exposure significantly reduced the eNOS dimer/monomer ratio in MAs from male offspring, but not females (Figure 4A), confirming sex-specific eNOS uncoupling. BH4, an essential cofactor for eNOS coupling, was significantly reduced in MAs from male, but not female, CINA offspring (Figure 4B). Consistent with impaired BH4 biosynthesis, protein expression of GTPCH1, the rate-limiting enzyme in de novo BH4 synthesis, was significantly downregulated in male (Figure 4C), but not female (Figure 4D) CINA exposed offspring. These data identify a sex-specific impairment of the BH4–eNOS axis as a key mechanism underlying vascular dysfunction in male offspring.

Figure 4. Effect of prenatal CINA exposure on eNOS dimer/monomer, BH4 levels, and GTPCH1 protein in mesenteric arteries (MAs).

A, Low-temperature SDS-PAGE immunoblots showing eNOS dimers and monomers in MAs from male and female offspring, with quantification of the eNOS dimer-to-monomer ratios (n=6 offspring per sex per group). B, Total BH4 levels in MAs from male and female offspring (n=5 offspring per sex per group). C and D, Representative immunoblots and quantification of GTPCH1 protein expression in male (C; n=6 offspring per group) and female (D; Saline, n=5; CINA, n=6) offspring. Data are presented as mean ± SEM. Unpaired two-tailed Student’s t-test was used for all comparisons. Normality was assessed by the Shapiro–Wilk test, and homogeneity of variance by the F test. ns, no significant.

5. Functional significance of BH4 deficiency in prenatal CINA-mediated NO deficiency and hypertensive programming

To directly assess the functional role of BH4 deficiency, rescue experiments were performed using exogenous BH4 supplementation. Prenatal CINA exposure significantly reduced endothelial NO production in MA sections from male offspring, as assessed by 4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate (DAF-FM DA) fluorescence (Figure 5A and B), with no effect in females (Figure S2A and B). BH4 treatment (10 μM) fully restored NO production in male CINA offspring to control levels (Figure 5B).

Figure 5. BH4 supplementation restores nitric oxide production and vascular function in CINA-exposed male offspring.

A, Representative DAF-FM fluorescence images of NO production in MAs in the absence or presence of BH4 (10 μM). Scale bar, 50 μm. B, Quantification of endothelial DAF-FM fluorescence intensity (n=6 offspring per group). C and D, Ach-induced relaxation in MAs from saline (C; n=9 offspring) and CINA (D; n=7 offspring) male offspring in the absence or presence of BH4 (10 μM). E, Ach-induced relaxation in saline (n=7 offspring) and CINA (n=9 offspring) male offspring in the presence of BH4. One vessel ring was obtained from each offspring, and the same animals were used across all concentrations. F, Ang II (100 ng/kg)-induced ΔMAP in male offspring in the absence or presence of BH4 (10 mg/kg) (n=6–7/group). Data are presented as mean ± SEM; n represents the number of offspring per group. Panel B was analyzed using an unpaired two-tailed Student’s t test. Panels C–E were analyzed using two-way repeated-measures ANOVA, with Ach concentration as the within-subjects factor and treatment condition as the between-subjects factor. The Greenhouse–Geisser correction was applied, followed by Bonferroni multiple-comparison test.. Panel F was analyzed using two-way ANOVA, with prenatal exposure and BH4 treatment as factors, followed by Bonferroni post hoc tests. Normality was assessed using the Shapiro-Wilk test (B-F), and homogeneity of variance was assessed using the F test (B) or Brown-Forsythe test (C-F). *P<0.05 and **P<0.01 vs. corresponding control; ns, no significance.

Using pressure myography, BH4 supplementation had no effect on Ach-induced vasodilation in saline control males (Figure 5C), but significantly improved vasorelaxation in male CINA offspring (Figure 5D), eliminating group differences (Figure 5E). In vivo, Ang II–induced ΔMAP responses were significantly higher in male CINA offspring than controls; acute BH4 administration significantly attenuated these responses and abolished group differences (Figure 5F). These results establish BH4 deficiency as a key mediator of prenatal CINA-induced vascular dysfunction and hypertensive programming.

6. Epigenetic regulation of prenatal CINA exposure-mediated eNOS uncoupling via FTO-dependent m6A demethylation

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that prenatal CINA exposure did not alter eNOS or GTPCH1 mRNA levels in male or female offspring (Figure S3A and B), indicating post-transcriptional regulation. Given the role of m6A modification in translational control,^28,32 potential m6A sites were examined. Sequence-based RNA adenosine methylation site predictor (SRAMP)⁴² analysis identified predicted six high-confidence m6A sites in rat eNOS mRNA at positions 389, 1486, 2373, 3049, 3111, and 3947(Figure 6A, left panel), whereas no high-confidence sites were identified in GTPCH1 mRNA (Figure S4). To functionally validate m6A involvement, we performed m6A-specific RNA immunoprecipitation (MeRIP) followed by qRT-PCR (MeRIP-qPCR) on male offspring MAs m6A levels at 3’UTR of eNOS mRNA (Figure 6A, right panel). MeRIP-qPCR demonstrated selective reduction of m6A enrichment at the 3′UTR site (position 3947) of eNOS mRNA in male CINA offspring (Figure 6 A, right panel). Correspondingly, FTO protein expression was significantly increased in male, but not female, CINA offspring (Figure 6B), while other m6A regulators remained unchanged (Figure S5).

Figure 6. FTO inhibition normalizes eNOS and GTPCH1 protein expression in mesenteric arteries from CINA-exposed male offspring.

A, Schematic representation of rat eNOS mRNA (NM_021838.2) showing predicted high-confidence m6A sites within the coding sequence (CDS) and 3′UTR (left). Site-specific m6A enrichment of eNOS mRNA assessed by MeRIP-qPCR (Right; n=4 pooled samples/group, with 3 rats per pool). Saline and CINA groups were compared at each site. Normality was assessed by the Shapiro–Wilk test. All sites except eNOS 3049 met parametric assumptions and were analyzed using unpaired two-tailed Student’s t-test, with homogeneity of variance assessed using the F test. The eNOS 3049 site was analyzed using the Mann–Whitney U test. No multiplicity adjustment was applied across the different sites. B, Representative immunoblots and quantification of FTO protein in MAs from male (Saline, n=6; CINA, n=6) and female (Saline, n=6; CINA, n=5) offspring. C, eNOS and FTO protein expression in organ-cultured MAs from saline and CINA male offspring treated with treated with vehicle (DMSO, 48 h). D, Correlation between FTO and eNOS protein levels in male offspring MAs shown in C (pearson correlation coefficient). Normality was assessed using the Shapiro–Wilk test. E, eNOS protein levels in organ-cultured MAs from saline and CINA male offspring following treatment with the FTO inhibitor FB23-2 (10 μM, 48 h) (n=5 offspring per group). F, GTPCH1 protein expression in organ-cultured MAs from saline and CINA male offspring following 48 h treatment with vehicle (left; saline, n=6; CINA, n=5) or FB23-2 (right; 10 μM, 48 h; n=5 offspring per group). GAPDH or β-actin served as loading controls. Data are presented as means ± SEM. For panels B, C, E, and F, normality was assessed using the Shapiro–Wilk test and homogeneity of variance using the F test; all data met parametric assumptions and were analyzed using unpaired two-tailed Student’s t-test. ns, no significant.

To investigate the potential mechanistic linkage between FTO/m6A methylation and eNOS protein expression, we used an ex vivo mesenteric artery (MA) culture model to treat with or without the specific FTO inhibitor FB23-2⁴³ (10 μM) for 48 hours. Our data showed in the absence of FB23-2 (Figure 6C) that protein levels of both eNOS and FTO in the ex vivo cultured MAs were significantly higher in CINA exposed group than the saline control group. Ex vivo MA culture experiments revealed a strong positive correlation between FTO and eNOS protein levels (Figure 6D). Pharmacologic inhibition of FTO with FB23-2 normalized eNOS (Figure 6E). Additionally, CINA exposure significantly decreased GTPCH1 protein expression in the cultured MAs (Figure 6F, left panel), which was reversed by FB23-2 treatment (Figure 6F, right panel). Furthermore, pharmacological inhibition of FTO with FB23-2 also restored CINA-mediated eNOS dimerization (Figure S6A). The CINA exposure-mediated decreased NO production (Figure S6B) was normalized by FB23-2 treatment (Figure S6C) in the cultured MAs. These data demonstrate a causal role for FTO-mediated m6A demethylation in prenatal CINA-induced eNOS uncoupling.

7. FTO-mediated m6A demethylation drives nicotine-induced eNOS uncoupling in human endothelial cells

To establish a human-relevant mechanism, human umbilical vein endothelial cells (HUVECs) were treated with nicotine (10⁻⁶ M, 24 h). Our data show that nicotine exposure significantly increased eNOS protein levels (Figure S7A), reduced GTPCH1 protein levels (Figure S7Fi), decreased NO production (Figure S8A and B), and increased DHE-detectable superoxide-dependent signal generation in an L-NAME–sensitive manner (Figure S9). Additionally, BH4 supplementation restored NO production (Figure S8C and D).

Next, we examined nicotine’s impact on site-specific m6A levels in eNOS mRNA in HUVECs. Using the SRAMP algorithm, we found a high-confidence m6A site in the 3′UTR (position 4320) and another in the CDS (position 3225) of human eNOS mRNA (Figure S7B, left panel). MeRIP-qPCR revealed that nicotine treatment selectively reduced m6A enrichment at a high-confidence 3′UTR site (4320) of human eNOS mRNA (Figure S7B, right panel). This pattern mirrored observations in vessels from nicotine-exposed male rat offspring.

To determine if FTO-derived m6A signaling directly regulates eNOS and GTPCH1, HUVECs were treated with either the specific FTO inhibitor FB23-2 or FTO-targeting siRNA (10 nM). Our data showed that FTO siRNA effectively knocked down FTO protein levels (Figure S7C, panel 1 and S7Fii) and resulted in decreased eNOS protein levels (Figure S7C, panel 1) but increased GTPCH1 protein levels (Figure S7Fii) in HUVECs. Furthermore, knockdown of FTO with siRNA reversed the nicotine-mediated changes of eNOS protein (Figure S7C, panel 2). Similarly, pharmacologic inhibition of FTO with FB23-2 produced dose-dependent decreases in eNOS protein levels (Figure S7D, left panel) and increases in GTPCH1 protein levels (Figure S7Fiii) in HUVECs. Critically, FB23-2 treatment (5 μM) abolished the nicotine-induced alterations in eNOS (Figure S7D, right panel) and GTPCH1 protein levels (Figure S7Fiv). Additionally, nicotine significantly reduced the eNOS dimer/monomer ratio in control cells treated with scrambled siRNA (Figure S7E). However, treatment with FTO siRNA increased the dimer/monomer ratio under both basal and nicotine-exposed conditions and eliminated the difference between nicotine-treated and control groups (Figure S7E) in HUVECs. Functionally, our data showed that inhibition of FTO (m6A demethylase) activity with FB23-2 rescued the nicotine-induced reduction of NO production in HUVECs (Figure S10). These findings confirm that FTO-mediated m6A demethylation is a conserved mechanism linking nicotine exposure to endothelial dysfunction.

Discussion

The present study demonstrates that prenatal exposure to CINA, modeling maternal e-cigarette vaping, results in programming of long-lasting and sex-dependent vascular dysfunction in adult male offspring. Extending our recent findings that prenatal CINA exposure selectively upregulates the RNA demethylase FTO and NOX2-dependent oxidative stress in male offspring vasculature,³⁷ we provide new evidence that disruption of the BH4–eNOS coupling axis represents an important mechanism contributing to endothelial dysfunction and enhanced pressor responsiveness. Specifically, the main findings are that prenatal CINA exposure: (i) selectively enhances vasoconstrictor-induced blood pressure responses and impairs endothelium-dependent vasodilation in male offspring; (ii) induces paradoxical upregulation of vascular eNOS protein that is functionally uncoupled, resulting in reduced NO bioavailability and excessive reactive oxygen species (ROS) generation; (iii) disrupts tetrahydrobiopterin (BH4) homeostasis via downregulation of GTPCH1, leading to impaired eNOS dimerization; and (iv) identifies FTO-dependent m6A RNA demethylation as a novel epigenetic mechanism driving nicotine-induced eNOS uncoupling. Together, these findings establish a mechanistic framework linking prenatal nicotine exposure to sex-specific vascular programming associated with increased hypertensive susceptibility through epitranscriptomic regulation of endothelial redox signaling.

Prenatal nicotine exposure programs a latent hypertensive phenotype

A central and clinically relevant observation of the present study is that prenatal CINA exposure did not alter baseline arterial blood pressure in adult offspring, but markedly exaggerated blood pressure responses to vasoconstrictors and impaired endothelium-dependent vasodilation in males. This dissociation between resting blood pressure and stress responsiveness parallels epidemiological findings showing that offspring exposed to maternal smoking during gestation often remain normotensive in early adulthood but exhibit increased susceptibility to hypertension and cardiovascular disease later in life.^2,3,6,7 These findings support the concept that developmental insults may not immediately elevate basal blood pressure but instead compromise vascular adaptability, lowering the threshold for disease when homeostatic reserve is challenged.

From a physiological perspective, endothelial NO signaling plays a critical role in buffering vasoconstrictor stimuli and maintaining blood pressure stability.^10,12 The impaired Ach-induced hypotensive responses and exaggerated Ang II pressor responses observed in male CINA offspring indicate a loss of this buffering capacity. Importantly, NOS inhibition abolished group differences in Ach-induced hypotensive responses, directly implicating endothelial NO signaling as a principal target of prenatal nicotine exposure. Thus, prenatal nicotine exposure establishes a compensated vascular state in which endothelial dysfunction is masked at rest but becomes unmasked during neurohumoral or vasoactive stress, a hallmark of early cardiovascular programming.

Sex specificity and developmental susceptibility

The vascular phenotype induced by prenatal CINA exposure was strictly male-specific. Female offspring exhibited preserved endothelial function, normal BH4 levels, and intact NO signaling. This sexual dimorphism is consistent with a large body of developmental programming literature demonstrating greater vulnerability of males to prenatal insults affecting cardiovascular, metabolic, and neuroendocrine systems.^7,44,45,46 In contrast, the finding that female offspring were protected from prenatal CINA exposure-induced endothelial dysfunction, is consistent with a potential protective role of estrogen signaling, as previous studies have shown that estrogen enhances eNOS activity, supports BH4 biosynthesis, suppresses oxidative stress, and improves endothelial repair capacity.^47,48,49 We acknowledge, however, that the direct causal involvement of estrogen in the female-protective phenotype remains to be experimentally validated in this model, and this represents a limitation of the present study. Future studies employing ovariectomy, estrogen supplementation, or estrogen receptor blockade would be required to definitively test this hypothesis.

Our finding that prenatal nicotine exposure programs a sex-specific reduction in vascular GTPCH1/BH4, leading to eNOS uncoupling and endothelial dysfunction exclusively in male offspring, is strongly corroborated by recent genetic models of acute BH4 deficiency. Chuaiphichai et al.⁵⁰ demonstrated that inducible, endothelial-specific deletion of Gch1(the gene encoding GTPCH1) in adult mice recapitulates a profound sexual dimorphism: it causes progressive hypertension and severe NO-mediated vasodilatory impairment in males but only a mild effects in females. These independent data reinforce two fundamental principles: first, the BH4-eNOS axis is intrinsically more vulnerable to disruption in the male vasculature; second, the female vasculature possesses a remarkable resilience, likely mediated by estrogenic tone, which buffers against both acquired genetic deficiency and, as we show, developmental programming by environmental toxins. While genetic models establish BH4 deficiency as sufficient to induce male-predominant hypertension, our study identifies an upstream, environmentally triggered epitranscriptomic mechanism (FTO-m6A signaling) that installs this deficiency during development. Together, these parallel lines of evidence underscore that sex-specific vulnerability in the BH4 pathway is a fundamental principle in vascular biology, manifesting across both acquired genetic and developmental environmental etiologies.

Paradoxical eNOS upregulation with impaired NO bioavailability reveals eNOS uncoupling as a central mechanism

Despite impaired Ach-induced vasodilation and reduced NO bioavailability, our findings showed that male CINA-exposed offspring exhibited paradoxical upregulation of eNOS protein in resistance arteries. This dissociation between eNOS abundance and function is a hallmark of eNOS uncoupling, a pathological state in which eNOS generates more superoxide and hydrogen peroxide rather than NO.^{12,16,17,51,52,53,54} Consistent with this mechanism, prenatal CINA exposure reduced vascular nitrate/nitrite (NO_X) levels while markedly increasing endothelial superoxide and hydrogen peroxide production. These findings extend previous reports linking perinatal nicotine exposure to endothelial dysfunction by identifying eNOS uncoupling, rather than reduced eNOS abundance, as the primary defect.⁵⁵ Importantly, our present findings showed that vasodilation in response to the exogenous NO donor sodium nitroprusside remained intact. This suggests that vascular smooth muscle sensitivity to NO was preserved, confirming that the dysfunction resides upstream at the level of endothelial redox signaling. Moreover, the observation that PEG-catalase normalized vasodilatory responses further supports a causal role for hydrogen peroxide in mediating impaired endothelial function in this model. These data indicate that prenatal nicotine exposure does not diminish eNOS expression but instead converts eNOS into a pro-oxidant enzyme, fundamentally reprogramming endothelial redox signaling.

BH4 deficiency and GTPCH1 repression drive eNOS uncoupling

Mechanistically, we identify tetrahydrobiopterin (BH4) deficiency as the critical determinant of eNOS uncoupling in male offspring. BH4 is essential for stabilizing the eNOS dimer and ensuring efficient electron transfer during NO synthesis. Deficiency of BH4 leads to eNOS monomerization and diversion of electron flow toward superoxide production, a mechanism implicated in hypertension, diabetes, and atherosclerosis.^40,41,53 In line with this, our present study showed that prenatal CINA exposure significantly reduced BH4 levels and decreased the eNOS dimer-to-monomer ratio. We also observed that prenatal CINA exposure reduced the protein expression of GTPCH1, the rate-limiting enzyme for de novo BH4 biosynthesis, in male but not female mesenteric arteries. This primary synthetic defect creates an absolute shortage of BH4.

The causal importance of this pathway is supported by genetic models showing that endothelial-specific GCH1 deletion induces endothelial dysfunction and exaggerated Ang II responses, mirroring key aspects of our phenotype.^40,41,56 Importantly, our findings demonstrated that functional rescue with exogenous BH4 fully restored NO production, normalized endothelial vasodilation, and abolished exaggerated Ang II-induced pressor responses in prenatal CINA-exposed male offspring. These findings reveal that BH4 deficiency is the dominant limiting factor in prenatal CINA-induced programmed phenotype.

FTO-dependent m6A demethylation links prenatal nicotine exposure to endothelial reprogramming

A novel finding of the present study is the identification of RNA methylation as a proximal regulatory node linking prenatal nicotine exposure to long-term programming of endothelial dysfunction. m6A modification influences mRNA splicing, stability, translation efficiency, and ribonucleoprotein complex assembly.^25,27,57 FTO functions as a demethylase that removes m6A marks in a site- and context-dependent manner. We demonstrate that prenatal CINA exposure selectively increases FTO expression in male mesenteric arteries and reduces m6A methylation within eNOS 3′UTR. Reduced m6A can enhance translational efficiency by altering YTH-domain reader protein recruitment or ribosome loading, providing a mechanistic explanation for increased eNOS protein despite unchanged transcript levels. We acknowledge, however, that direct evidence for enhanced translation via polysome profiling or m6A reporter assays was not obtained in this study, and the precise molecular details linking site-specific 3′UTR hypomethylation to eNOS protein elevation remain to be resolved. Simultaneously, GTPCH1 protein is reduced without changes in mRNA abundance, and no high-confidence m6A site was identified in GTPCH1 transcripts. These findings suggest that FTO-mediated m6A remodeling may influence GTPCH1 through indirect mechanisms, including regulation of mRNAs encoding transcriptional regulators or RNA-binding proteins that control GTPCH1 translation, or through alterations in protein stability under conditions of increased oxidative stress. Notably, GTPCH1 has been reported to be susceptible to oxidative degradation,^20,41 raising the possibility that FTO-driven redox imbalance may further reduce GTPCH1 protein abundance.

Taken together, the combination of increased eNOS protein expression and reduced GTPCH1 protein expression could create an imbalance between eNOS and its essential cofactor BH4, thereby promoting eNOS uncoupling. These findings suggest that FTO may contribute to the coordinated regulation of endothelial redox homeostasis by influencing both eNOS abundance and BH4 availability.

Pharmacological inhibition or silencing of FTO normalized eNOS and GTPCH1 protein levels, restored dimerization, and reduced ROS production, establishing FTO as a necessary upstream mediator. We note, however, that FB23-2, while widely used as a selective FTO inhibitor, may have off-target effects at higher concentrations, and we did not perform mesenteric artery-specific FTO knockdown or overexpression in ex vivo or in vivo. The convergence of independent pharmacological and genetic approaches—in both rat vascular tissue and human endothelial cells—nevertheless provides complementary evidence supporting FTO as an upstream mediator. Given that m6A regulation is highly dynamic and responsive to environmental cues, FTO-dependent demethylation provides a plausible molecular memory mechanism by which transient prenatal nicotine exposure induces stable adult vascular phenotypes.

Convergence of FTO–NOX2 and FTO–eNOS pathways: a redox amplification network

Integrating our prior findings³⁷ with the current data reveals a coherent redox amplification network. FTO upregulation enhances NOX2-dependent ROS production while simultaneously promoting eNOS uncoupling through BH4 depletion. NOX2-derived superoxide can oxidize BH4 to BH2, further destabilizing eNOS dimers, while uncoupled eNOS itself becomes an additional ROS source. This establishes a self-reinforcing oxidative circuit that shifts endothelial signaling from NO dominance to ROS dominance. Such coordinated regulation suggests that FTO functions as an important epitranscriptomic regulator controlling endothelial redox tone. Rather than affecting a single pathway, prenatal nicotine exposure reprograms multiple enzymatic nodes converging on NO bioavailability and oxidative stress balance.

Translational relevance and conservation of mechanism in human endothelial cells

The translational significance of this pathway is reinforced by parallel findings in HUVECs, where nicotine exposure recapitulated the in vivo phenotype, including increased eNOS protein associated with reduced m6A level, reduced GTPCH1 levels, impaired NO production, enhanced ROS generation, and reduced eNOS dimerization. Both genetic silencing (siRNA knockdown of FTO) and pharmacological inhibition of FTO reversed these effects, demonstrating conservation of the FTO–m6A–eNOS axis across species. We acknowledge that HUVECs are venous in origin and may not fully replicate the biology of arterial endothelial cells; nevertheless, the consistent recapitulation of the core pathway in both rat mesenteric arteries and human venous endothelial cells, across distinct species and vessel types, suggests that this epitranscriptomic mechanism may operate across multiple endothelial cell types. Validation in human arterial endothelial cells or arterial tissue would further strengthen the translational relevance of these findings.

Given the widespread perception of e-cigarettes as a safer alternative to traditional smoking, our study raises significant concerns regarding vascular consequences of nicotine exposure during pregnancy. Our data suggest that nicotine itself, independent of combustion products, is sufficient to epigenetically program long-term endothelial dysfunction when exposure occurs during a critical window of fetal development. Furthermore, identification of a sex-specific epigenetic mechanism provides a plausible explanation for differential cardiovascular risk trajectories between male and female offspring.

Limitations and future directions

Several limitations should be acknowledged. First, while the present study focused on resistance arteries critical for blood pressure regulation, future studies should examine whether similar mechanisms operate in other vascular beds. Second, we acknowledge that our sex-stratified analytical approach does not directly test the sex-by-treatment interaction. Although the observed direction and/or magnitude of the CINA-associated effects differed between male and female offspring, these findings should not be interpreted as evidence of a statistically significant sex-by-treatment interaction. Formal testing of the sex-by-treatment interaction in future studies will be important to further establish the statistical significance and biological basis of the observed sex-specific responses. Third, although DHE fluorescence has been widely used to assess vascular oxidative stress, it may reflect multiple oxidation products. Therefore, our findings should be interpreted as changes in superoxide-dependent DHE fluorescence rather than absolute quantification of superoxide levels. Finally, while FTO-dependent m6A regulation was identified as a key mechanism, additional RNA-binding proteins and translational regulators may contribute to the observed phenotype.

Future studies should explore whether targeting FTO or restoring BH4 bioavailability during critical developmental windows can prevent nicotine-induced cardiovascular programming. Longitudinal studies examining susceptibility to hypertension and cardiovascular disease later in life will also be essential.

Perspectives

This study identifies a novel epitranscriptomic mechanism by which prenatal nicotine exposure results in programming of sex-specific vascular dysfunction later in life. Prenatal CINA exposure upregulates FTO, alters m6A methylation of eNOS mRNA, suppresses GTPCH1 and BH4 biosynthesis, and induces eNOS uncoupling—shifting endothelial signaling from NO-mediated vasoprotection toward oxidative stress and exaggerated pressor responses. Integrated with our prior identification of FTO-NOX2 signaling,³⁷ these findings suggest that FTO is an important epitranscriptomic regulator of endothelial redox homeostasis and reserve capacity. Targeting the FTO–BH4–eNOS axis may therefore represent a promising strategy for mitigating the long-term cardiovascular consequences of prenatal nicotine exposure.

Supplementary Material

• Expanded Materials & Methods

• Tables S1–S3

• Figure S1–S10

NOVELTY AND RELEVANCE.

What Is New?

Prenatal chronic intermittent nicotine aerosol (CINA) exposure programs sex-specific vascular dysfunction and increased hypertensive susceptibility in adult male offspring through endothelial nitric oxide synthase (eNOS) uncoupling. Prenatal CINA paradoxically increased eNOS protein expression while suppressing GTP cyclohydrolase-1 (GTPCH1) expression and tetrahydrobiopterin (BH4) bioavailability, leading to impaired eNOS dimerization, reduced nitric oxide bioavailability, and increased reactive oxygen species production. Mechanistically, increased expression of the m6A RNA demethylase FTO mediated these changes, whereas pharmacological inhibition or siRNA-mediated knockdown of FTO restored GTPCH1 expression, eNOS coupling, and nitric oxide production in resistance arteries and human endothelial cells.

What Is Relevant?

This study identifies FTO-dependent m6A RNA demethylation as a novel and potentially reversible epitranscriptomic mechanism linking prenatal nicotine exposure to programmed endothelial dysfunction and increased hypertensive susceptibility. Restoration of endothelial function by FTO inhibition suggests that developmental vascular programming may remain therapeutically modifiable in adulthood.

Clinical/Pathophysiological Implications

Maternal smoking and e-cigarette use during pregnancy remain important contributors to developmental cardiovascular risk. Our findings identify the FTO–GTPCH1–BH4–eNOS pathway as a previously unrecognized epitranscriptomic mechanism linking prenatal nicotine exposure to sex-specific endothelial dysfunction and increased hypertensive susceptibility. The restoration of eNOS coupling and nitric oxide bioavailability by FTO inhibition in adult vessels and human endothelial cells provides proof-of-concept that epitranscriptomic pathways may represent therapeutic targets after developmental programming has occurred. These findings provide a mechanistic foundation for developing strategies to prevent or mitigate endothelial dysfunction associated with adverse intrauterine nicotine exposure.

Nonstandard Abbreviations and Acronyms

Ach

acetylcholine

ALKBH5

ALKB homolog 5

Ang II

angiotensin II

BH4

tetrahydrobiopterin

BP

blood pressure

CINA

chronic intermittent nicotine aerosol

DAF-FM DA

4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate

DHE

dihydroethidium

eNOS

endothelial nitric oxide synthase

EC

endothelial cell

FTO

fat mass and obesity-associated protein

GTPCH1

GTP cyclohydrolase I

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

H₂O₂

hydrogen peroxide

HR

heart rate

HUVEC

human umbilical vein endothelial cell

L-NAME

Nω-nitro-L-arginine methyl ester

MAs

mesenteric arteries

m6A

N6-methyladenosine

METTL14

methyltransferase-like 14

METTL3

methyltransferase-like 3

NO

nitric oxide

NO_x

nitrite and nitrate

NOX2

NADPH oxidase 2

O₂^˙−

superoxide

PEG-catalase

catalase–polyethylene glycol

ROS

reactive oxygen species

WTAP

Wilms’ tumor 1-associating protein