Xuesi Max Shao

BACKGROUND: Prenatal nicotine exposure from maternal smoking or e-cigarette use increases cardiovascular risk in offspring, but the underlying mechanisms remain unclear. We tested whether FTO (fat mass and obesity-associated protein)-mediated N6-methyladenosine RNA demethylation drives eNOS (endothelial nitric oxide [NO] synthase) uncoupling and programmed hypertensive susceptibility following prenatal nicotine exposure. METHODS: Using a clinically relevant rat model of chronic intermittent nicotine aerosol exposure or saline aerosol control during gestational days 4 to 21, experiments were performed in adult offspring and human umbilical vein endothelial cells. RESULTS: Prenatal chronic intermittent nicotine aerosol 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 NO bioavailability, increased superoxide and hydrogen peroxide production, impaired eNOS dimerization, and decreased tetrahydrobiopterin levels associated with reduced GTPCH1 (GTP cyclohydrolase-1) expression, consistent with eNOS uncoupling. Mechanistically, prenatal chronic intermittent nicotine aerosol increased vascular FTO expression and induced site-specific N6-methyladenosine 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 human umbilical vein endothelial cells, 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 N6-methyladenosine RNA demethylation, leading to tetrahydrobiopterin deficiency, eNOS uncoupling, and vascular dysfunction in male offspring. The FTO—tetrahydrobiopterin—eNOS pathway represents a novel epitranscriptomic mechanism underlying programmed hypertensive susceptibility and a potential therapeutic target for offspring exposed to nicotine during development.

Electronic cigarettes (E-Cig) are a new way of delivering nicotine, gaining popularity among adolescents and young adults, who often do not realize their harmful effects. Although the adverse effects of E-Cigs on the liver and heart have been demonstrated, their effects on the skeletal muscle have not been well studied. In this study, we evaluated the skeletal muscle effects of E-Cig aerosol, delivered in a manner similar to human vaping, in a mouse model of obesity induced by a high-fat diet (HFD). C57BL/6 mice, fed either a normal chow diet (NCD) or HFD, were exposed to either saline aerosol control or aerosol generated from Blu PLUS^TM containing 0% or 2.4% nicotine for 12 weeks. Mice fed an NCD were included to distinguish whether E-Cig effects on the skeletal muscle required the presence of obesity induced by an HFD. The soleus muscle, an oxidative muscle rich in mitochondria, was assessed by Western blotting, electron microscopy, and biochemical assays. An NCD group was included to assess the baseline effects of HFD-induced obesity, on the skeletal muscle. The skeletal muscle from HFD-fed mice exposed to E-Cig 2.4% had reduced levels of phospho-AMPK compared with saline and E-Cig 0% groups, while E-Cigs had no effect on NCD-fed mice. Levels of phospho-adipose triglyceride lipase were also reduced in both E-Cig 2.4% and 0% compared with the saline group. These metabolic protein impairments were accompanied by increased levels of oxidative stress and phospho-p38 MAPK. Deregulation of the autophagy markers, microtubule-associated protein 1A/1B-light chain 3 (LC3-I; inactive form) and LC3-II (active form), was also observed, evidenced by decreased levels of LC3-II, ratio LC3-II/LC3-I, and increased levels of p62. Transmission electron microscopy analysis showed that E-Cig 2.4% induced damage to mitochondrial structure compared with the saline or E-Cig 0% groups. These findings suggest that E-Cig exposure on HFD impairs the skeletal muscle, adding to the growing list of affected organs for ongoing regulatory efforts concerning nicotine-containing substances.

Effects of electronic cigarettes (E-cigs) compared with tobacco cigarettes (T-cigs) in the Western population are largely undetermined. Here, we assessed chronic, intermittent exposures to either E-cigs or T-cigs on cardiovascular parameters in hyperlipidemic mice with similar nicotine levels. Our findings suggest that both E-cigs and T-cigs imposed adverse effects on cardiac function, heart chamber size, and atherosclerotic lesions in diet and sex-dependent manners, suggesting that electronic cigarettes are not a safer alternative to conventional cigarettes.

BACKGROUND: E-cigarette use increases the risk of blood pressure (BP) elevation in users, though the underlying mechanisms remain unclear. The mineralocorticoid receptor (MR) is known to play an important role in the regulation of renal electrolyte balance and BP, and is protected by 11β-HSD2 (11β-hydroxysteroid dehydrogenase type 2). However, little is known about the effects of renal MR on e-cigarette-induced BP elevation. METHODS: C57BL/6J male mice were exposed to aerosolized PBS, e-cigarettes without nicotine, and e-cigarettes with 2.4% nicotine, with concurrent exposure to either a vehicle or the MR antagonist eplerenone. RESULTS: Inhalation of e-cigarettes with nicotine markedly induced renal MR abundance and increased mean arterial BP in response to elevated plasma nicotine levels in C57BL/6J mice. Induction of MR by e-cigarettes was correlated with a reduction in 11β-HSD2 and activation of pSer ⁹ GSK3β (glycogen synthase kinase-3β phosphorylation) within the kidneys. In contrast, e-cigarettes increased the urinary ratio of corticosterone to 11-dehydrocorticosterone, reduced urinary sodium content, and elevated renal epithelial sodium channel expression. However, inhaling e-cigarettes without nicotine did not affect these metabolic parameters. Treatment with eplerenone normalized BP, reversed urinary metabolic profiles, and reduced epithelial sodium channel by inhibiting renal pSer ⁹ GSK3β in nicotine e-cigarette-exposed mice. In mouse renal Cortical Collecting Duct (CCD) M1 cells, aerosol nicotine from e-cigarettes increased MR while decreasing 11β-HSD2, and these effects are likely via activation of pSer ⁹ GSK3β through a nicotinic receptor-mediated mechanism. CONCLUSIONS: Our findings highlight the potential renal health damage from e-cigarettes and suggest that aerosol nicotine-mediated induction of MR action in the kidneys may contribute to electronic smoking-induced BP elevation.

BACKGROUND: Cigarette smoking/nicotine exposure in pregnancy shows an increased risk of hypertension in offspring, but the mechanisms are unclear. This study tested the hypothesis that m6A RNA hypomethylation epigenetically regulates vascular NOX (NADPH oxidase) and reactive oxygen species production, contributing to the fetal programming of a hypertensive phenotype in nicotine-exposed offspring. METHODS: Pregnant rats were exposed to episodic chronic intermittent nicotine aerosol (CINA) or saline aerosol control from gestational day 4 to day 21, and experiments were performed in 6-month–old adult offspring. RESULTS: Antenatal CINA exposure augmented Ang II (angiotensin II)–stimulated blood pressure response in male, but not female offspring. Moreover, CINA increased vascular NOX2 expression and superoxide production exclusively in male offspring. Inhibition of NOX2 with gp91ds-tat, both ex vivo and in vivo, mitigated the CINA-induced elevation in superoxide production and blood pressure response. Notably, CINA enhanced the expression of vascular m6A demethylase FTO (fat mass and obesity-associated protein), while reducing the total vascular m6A abundance and specific m6A methylation of the NOX2 gene. Additionally, ex vivo inhibition of FTO with FB23-2 attenuated CINA-induced increases in vascular NOX2 expression. In vitro experiments using human umbilical vein endothelial cells demonstrated that nicotine dose-dependently upregulated FTO and NOX2 protein abundance, which were reversed by treatment with the FTO inhibitor FB23-2 or FTO knockdown using siRNAs. CONCLUSIONS: This study uncovers a new mechanism: m6A demethylase FTO-mediated epigenetic upregulation of vascular NOX2 signaling in CINA-induced hypertensive phenotype. This insight could lead to a therapeutic target for preventing and treating developmental hypertension programming.

Background: Anxiety disorders are the most prevalent mental illnesses in the U.S. and are estimated to consume one-third of the country’s mental health treatment cost. Although anxiolytic therapies are available, many patients still exhibit treatment resistance, relapse, or substantial side effects. Further, due to the COVID-19 pandemic and stay-at-home order, social isolation, fear of the pandemic, and unprecedented times, the incidence of anxiety has dramatically increased. Previously, we have demonstrated dihydromyricetin (DHM), the major bioactive flavonoid extracted from Ampelopsis grossedentata, exhibits anxiolytic properties in a mouse model of social isolation-induced anxiety. Because GABAergic transmission modulates the immune system in addition to the inhibitory signal transmission, we investigated the effects of short-term social isolation on the neuroimmune system. Methods: Eight-week-old male C57BL/6 mice were housed under absolute social isolation for 4 weeks. The anxiety-like behaviors after DHM treatment were examined using elevated plus-maze and open field behavioral tests. Gephyrin protein expression, microglial profile changes, NF-κB pathway activation, cytokine level, and serum corticosterone were measured. Results: Socially isolated mice showed increased anxiety levels, reduced exploratory behaviors, and reduced gephyrin levels. Also, a dynamic alteration in hippocampal microglia were detected illustrated as a decline in microglia number and overactivation as determined by significant morphological changes including decreases in lacunarity, perimeter, and cell size and increase in cell density. Moreover, social isolation induced an increase in serum corticosterone level and activation in NF-κB pathway. Notably, DHM treatment counteracted these changes. Conclusion: The results suggest that social isolation contributes to neuroinflammation, while DHM has the ability to improve neuroinflammation induced by anxiety.

Maternal e-cigarette (e-cig) exposure is a pressing perinatal health concern. Emerging evidence reveals its potential adverse impacts on brain development in offspring, yet the underlying mechanisms are poorly understood. The present study tested the hypothesis that fetal e-cig exposure induces an aberrant DNA methylation profile in the developing brain, leading to alteration of autophagic flux signaling and programming of a sensitive phenotype to neonatal hypoxic-ischemic encephalopathy (HIE). Pregnant rats were exposed to chronic intermittent e-cig aerosol. Neonates were examined at the age of 9 days old. Maternal e-cig exposure decreased the body weight and brain weight but enhanced the brain-to-body weight ratio in the neonates. E-cig exposure induced a gender-dependent increase in hypoxic-ischemia-induced brain injury in male neonates associated with enhanced reactive oxygen species (ROS) activity. It differentially altered DNA methyltransferase expression and enhanced both global DNA methylation levels and specific CpG methylation at the autophagy-related gene 5 (ATG5) promoter. In addition, maternal e-cig exposure caused downregulations of ATG5, microtubule-associated protein 1 light chain 3β, and sirtuin 1 expression in neonatal brains. Of importance, knockdown of ATG5 in neonatal pups exaggerated neonatal HIE. In conclusion, the present study reveals that maternal e-cig exposure downregulates autophagy-related gene expression via DNA hypermethylation, leading to programming of a hypoxic-ischemic sensitive phenotype in the neonatal brain.

Whether SLC4A11 transports ammonia and its potential mode of ammonia transport ([ ], NH₃, or NH₃-2H⁺ transport have been proposed) are controversial. In the absence of ammonia, whether SLC4A11 mediates significant conductive H⁺(OH⁻) transport is also controversial. The present study was performed to determine the mechanism of human SLC4A11 ammonia transport and whether the transporter mediates conductive H⁺(OH⁻) transport in the absence of ammonia. We quantitated H⁺ flux by monitoring changes in intracellular pH (pH_i) and measured whole cell currents in patch-clamp studies of HEK293 cells expressing the transporter in the absence and presence of NH₄Cl. Our results demonstrate that SLC4A11 mediated conductive H⁺(OH⁻) transport that was stimulated by raising the extracellular pH (pH_e). Ammonia-induced HEK293 whole cell currents were also stimulated by an increase in pH_e. In studies using increasing NH₄Cl concentrations with equal [ ] extracellular and intracellular concentrations, the shift in the reversal potential ( E_rev) due to the addition of ammonia was compatible with NH₃-H⁺ transport competing with H⁺(OH⁻) rather than NH₃-nH⁺ ( n ≥ 2) transport. The increase in equivalent H⁺(OH⁻) flux observed in the presence of a transcellular H⁺ gradient was also compatible with SLC4A11-mediated NH₃-H⁺ flux. The NH₃ versus E_rev data fit a theoretical model suggesting that NH₃-H⁺ and H⁺(OH⁻) competitively interact with the transporter. Studies of mutant SLC4A11 constructs in the putative SLC4A11 ion coordination site showed that both H⁺(OH⁻) transport and ammonia-induced whole cell currents were blocked suggesting that the H⁺(OH⁻) and NH₃-H⁺ transport processes share common features involving the SLC4A11 transport mechanism.