Maternal Electronic Cigarette Exposure Induces Dysregulation of Autophagy via Oxidative Stress/DNA Methylation in Pulmonary Hypertension Offspring

Abstract

Objective

Electronic cigarettes (ECs) differ from traditional tobacco smoke but may contribute to cardiopulmonary remodeling. Pulmonary hypertension (PH), characterized by pulmonary artery and right ventricle remodeling, poses a significant risk of mortality in infants, children, and adolescents. However, the impact of maternal EC exposure on PH development in offspring remains unclear. To address this, we established a PH rat model with maternal EC exposure.

Methods

Maternal EC exposure was initiated on gestation day 12 via electronic nicotine delivery systems. Offspring were administered monocrotaline (MCT) at 6 weeks of age (6-wo) to induce PH. Mechanistic experiments were conducted at 10-week-old (10-wo). Protein expression of NADPH oxidases, DNA methyltransferases, and autophagy-related markers was analyzed by Western blot. Morphological changes and the severity of PH were evaluated via hematoxylin and eosin (HE) staining and echocardiography, respectively. Furthermore, the involvement of the oxidative stress/DNA methylation/autophagy axis in response to maternal EC exposure was confirmed through a combination of ELISA, Western blot, HE staining, and echocardiography. Additionally, ATG5 mRNA expression was measured by qRT-PCR.

Results

Compared with control conditions, maternal EC exposure significantly worsened MCT-induced PH in male offspring. This was associated with increased oxidative stress, DNA hypomethylation, and anomalous autophagy in the offspring. In vivo treatment with chloroquine inhibited autophagy and ameliorated PH development in offspring exposed to maternal EC. Furthermore, N-acetylcysteine (NAC), an antioxidant, attenuated maternal EC exposure-induced oxidative stress, DNA hypomethylation, and excessive autophagy, thereby improving PH. DNA hypermethylation also reversed PH development, accompanied by reduced oxidative stress and suppressed autophagy. ATG5, a key regulator of autophagy, was identified as a potential therapeutic target, as its repression mitigated PH in maternal EC-exposed offspring.

Conclusion

Maternal EC exposure induces oxidative stress and DNA hypomethylation in offspring, leading to anomalous autophagy and exacerbation of PH development. Targeting ATG5-mediated autophagy may represent a novel therapeutic approach for improving PH outcomes in offspring exposed to maternal EC.

Pregnant rats were exposed to either EC vapor or standard air from gestation day 12 until 2 days before delivery, with all offspring undergoing PH induction at 6-wo. Offspring exposed to maternal EC presented increased oxidative stress, which in turn affected DNA methylation patterns. The decreased DNA methylation in male offspring led to the activation of autophagy, exacerbating the development of PH. Treatment with ATG5 siRNA inhibited autophagy and alleviated heightened PH in male offspring with maternal EC exposure.

Keywords

Electronic cigarette / Maternal / Pulmonary hypertension / Offspring / Oxidative stress / DNA methylation / Autophagy

References

[1] Zhu SH, Sun JY, Bonnevie E, et al. Four hundred and sixty brands of e-cigarettes and counting: implications for product regulation. Tob Control. 2014;23 Suppl 3(Suppl 3):iii3–9.

[2] Cheng T. Chemical evaluation of electronic cigarettes. Tob Control. 2014;23 Suppl 2(Suppl 2):ii11–17.

[3] KosmiderL, SobczakA, FikM, et al.. Carbonyl compounds in electronic cigarette vapors: effects of nicotine solvent and battery output voltage. Nicotine Tob Res., 2014, 16(10): 1319-1326.

[4] MikheevVB, BrinkmanMC, GranvilleCA, et al.. Real-time measurement of electronic cigarette aerosol size distribution and metals content analysis. Nicotine Tob Res., 2016, 18(9): 1895-1902.

[5] MaessenGC, WijnhovenAM, NeijzenRL, et al.. Nicotine intoxication by e-cigarette liquids: a study of case reports and pathophysiology. Clin Toxicol (Phila)., 2020, 58(1): 1-8.

[6] GetiyeY, PetersonMR, PhillipsBD, et al.. E-cigarette exposure with or without heating the e-liquid induces differential remodeling in the lungs and right heart of mice. J Mol Cell Cardiol., 2022, 168: 83-95.

[7] SchweitzerKS, ChenSX, LawS, et al.. Endothelial disruptive proinflammatory effects of nicotine and e-cigarette vapor exposures. Am J Physiol Lung Cell Mol Physiol., 2015, 309(2): L175-187.

[8] HansmannG. Pulmonary hypertension in infants, children, and young adults. J Am Coll Cardiol., 2017, 69(20): 2551-2569.

[9] WangL, ZhangM, ChenX, et al.. Assessment of reversibility in pulmonary hypertension related to congenital heart disease by using biomarkers and clinical features. Congenital Heart Disease., 2022, 17(1): 87-97.

[10] KockenJMM, da Costa MartinsPA. Epigenetic regulation of pulmonary arterial hypertension-induced vascular and right ventricular remodeling: new opportunities?. Int J Mol Sci., 2020, 21238901.

[11] OmuraJ, SatohK, KikuchiN, et al.. ADAMTS8 promotes the development of pulmonary arterial hypertension and right ventricular failure: a possible novel therapeutic target. Circ Res., 2019, 125(10): 884-906.

[12] ThenappanT, ChanSY, WeirEK. Role of extracellular matrix in the pathogenesis of pulmonary arterial hypertension. Am J Physiol Heart Circ Physiol., 2018, 315(5): H1322-H1331.

[13] FriedND, MorrisTM, WhiteheadA, et al.. Angiotensin II type 1 receptor mediates pulmonary hypertension and right ventricular remodeling induced by inhaled nicotine. Am J Physiol Heart Circ Physiol., 2021, 320(4): H1526-H1534.

[14] WangQ, SundarIK, BlumJL, et al.. Prenatal exposure to electronic-cigarette aerosols leads to sex-dependent pulmonary extracellular-matrix remodeling and myogenesis in offspring mice. Am J Respir Cell Mol Biol., 2020, 63(6): 794-805.

[15] ZhangCF, ZhaoFY, XuSL, et al.. Autophagy in pulmonary hypertension: Emerging roles and therapeutic implications. J Cell Physiol., 2019, 234(10): 16755-16767.

[16] GuoL, LiY, TianY, et al.. eIF2alpha promotes vascular remodeling via autophagy in monocrotaline-induced pulmonary arterial hypertension rats. Drug Des Devel Ther., 2019, 13: 2799-2809.

[17] QipshidzeN, TyagiN, MetreveliN, et al.. Autophagy mechanism of right ventricular remodeling in murine model of pulmonary artery constriction. Am J Physiol Heart Circ Physiol., 2012, 302(3): H688-696.

[18] JinT, LuJ, LvQ, et al.. Farnesyl diphosphate synthase regulated endothelial proliferation and autophagy during rat pulmonary arterial hypertension induced by monocrotaline. Mol Med., 2022, 28194.

[19] OrnatowskiW, LuQ, YegambaramM, et al.. Complex interplay between autophagy and oxidative stress in the development of pulmonary disease. Redox Biol., 2020, 36. 101679

[20] Wu YC, Wang WT, Lee SS, et al. Glucagon-like peptide-1 receptor agonist attenuates autophagy to ameliorate pulmonary arterial hypertension through Drp1/NOX- and Atg-5/Atg-7/Beclin-1/LC3beta pathways. Int J Mol Sci. 2019;20(14):3435.

[21] LiL, TanJ, MiaoY, et al.. ROS and autophagy: interactions and molecular regulatory mechanisms. Cell Mol Neurobiol., 2015, 35(5): 615-621.

[22] Gonzalez-RodriguezP, CherayM, FullgrabeJ, et al.. The DNA methyltransferase DNMT3A contributes to autophagy long-term memory. Autophagy., 2021, 17(5): 1259-1277.

[23] ChenYC, LinIC, SuMC, et al.. Autophagy impairment in patients with obstructive sleep apnea modulates intermittent hypoxia-induced oxidative stress and cell apoptosis via hypermethylation of the ATG5 gene promoter region. Eur J Med Res., 2023, 28182.

[24] RichmondRC, Sillero-RejonC, KhoujaJN, et al.. Investigating the DNA methylation profile of e-cigarette use. Clin Epigenetics., 2021, 131183.

[25] Wills TA, Soneji SS, Choi K, et al. E-cigarette use and respiratory disorders: an integrative review of converging evidence from epidemiological and laboratory studies. Eur Respir J. 2021;57(1):1901815.

[26] YuW, ChenZ, LiY, et al.. In utero chronic intermittent nicotine aerosol exposure increases ischemic heart injury in adult offspring via programming of Angiotensin II receptor-derived TGFbeta/ROS/Akt signaling pathway. Reprod Toxicol., 2024, 128. 108650

[27] Shintani-ishidaK, SakaK, YamaguchiK, et al.. MDMA induces cardiac contractile dysfunction through autophagy upregulation and lysosome destabilization in rats. Biochim Biophys Acta., 2014, 1842(5): 691-700.

[28] GaoJ, ZhangX, YuM, et al.. Cognitive deficits induced by multi-walled carbon nanotubes via the autophagic pathway. Toxicology., 2015, 337: 21-29.

[29] ChenZ, GongL, ZhangP, et al.. Epigenetic down-regulation of Sirt 1 via DNA methylation and oxidative stress signaling contributes to the gestational diabetes mellitus-induced fetal programming of heart ischemia-sensitive phenotype in late life. Int J Biol Sci., 2019, 15(6): 1240-1251.

[30] XiaoD, HuangX, LiY, et al.. Antenatal antioxidant prevents nicotine-mediated hypertensive response in rat adult offspring. Biol Reprod., 2015, 93366.

[31] BarmanSA, LiX, HaighS, et al.. Galectin-3 is expressed in vascular smooth muscle cells and promotes pulmonary hypertension through changes in proliferation, apoptosis, and fibrosis. Am J Physiol Lung Cell Mol Physiol., 2019, 316(5): L784-L797.

[32] Force USPST, Krist AH, Davidson KW, et al. Interventions for tobacco smoking cessation in adults, including pregnant persons: US preventive services task force recommendation statement. JAMA. 2021;325(3):265–279.

[33] Toppila-SalmiS, LuukkainenAT, XuB, et al.. Maternal smoking during pregnancy affects adult onset of asthma in offspring: a follow up from birth to age 46 years. Eur Respir J., 2020, 5561901857.

[34] LiG, SaadS, OliverBG, et al.. Heat or burn? Impacts of intrauterine tobacco smoke and e-cigarette vapor exposure on the offspring’s health outcome. Toxics., 2018, 6343.

[35] BurrageEN, AboazizaE, HareL, et al.. Long-term cerebrovascular dysfunction in the offspring from maternal electronic cigarette use during pregnancy. Am J Physiol Heart Circ Physiol., 2021, 321(2): H339-H352.

[36] AslanerDM, AlghothaniO, SaldanaTA, et al.. E-cigarette vapor exposure in utero causes long-term pulmonary effects in offspring. Am J Physiol Lung Cell Mol Physiol., 2022, 323(6): L676-L682.

[37] LiG, ChanYL, WangB, et al.. E-cigarettes damage the liver and alter nutrient metabolism in pregnant mice and their offspring. Ann N Y Acad Sci., 2020, 1475(1): 64-77.

[38] LiG, ChanYL, NguyenLT, et al.. Impact of maternal e-cigarette vapor exposure on renal health in the offspring. Ann N Y Acad Sci., 2019, 1452(1): 65-77.

[39] WalayatA, LiY, ZhangY, et al.. Fetal e-cigarette exposure programs a neonatal brain hypoxic-ischemic sensitive phenotype via altering DNA methylation patterns and autophagy signaling pathway. Am J Physiol Regul Integr Comp Physiol., 2021, 321(5): R791-R801.

[40] FriedND, WhiteheadA, LazartiguesE, et al.. Ovarian hormones do not mediate protection against pulmonary hypertension and right ventricular remodeling in female mice exposed to chronic, inhaled nicotine. Am J Physiol Heart Circ Physiol., 2022, 323(5): H941-H948.

[41] Sanchez-GloriaJL, Martinez-OlivaresCE, Rojas-MoralesP, et al.. Anti-inflammatory effect of allicin associated with fibrosis in pulmonary arterial hypertension. Int J Mol Sci., 2021, 22168600.

[42] ParkE, SafdarZ. Pulmonary hypertension in women. Methodist Debakey Cardiovasc J., 2024, 20(2): 70-80.

[43] ChenZ, WangL, KeJ, et al.. Effects of estrogen in gender-dependent fetal programming of adult cardiovascular dysfunction. Curr Vasc Pharmacol., 2019, 17(2): 147-152.

[44] LiaoSX, SunPP, GuYH, et al.. Autophagy and pulmonary disease. Ther Adv Respir Dis., 2019, 131753466619890538.

[45] KrymskayaVP, SnowJ, CesaroneG, et al.. mTOR is required for pulmonary arterial vascular smooth muscle cell proliferation under chronic hypoxia. FASEB J., 2011, 25(6): 1922-1933.

[46] PasquierB. Autophagy inhibitors. Cell Mol Life Sci., 2016, 73(5): 985-1001.

[47] ShahandehN, ChowdharyH, MiddlekauffHR. Vaping and cardiac disease. Heart., 2021, 107(19): 1530-1535.

[48] RenX, LinL, SunQ, et al.. Metabolomics-based safety evaluation of acute exposure to electronic cigarettes in mice. Sci Total Environ., 2022, 839. 156392

[49] Correia-AlvarezE, KeatingJE, GlishG, et al.. Reactive oxygen species, mitochondrial membrane potential, and cellular membrane potential are predictors of e-liquid induced cellular toxicity. Nicotine Tob Res., 2020, 22(Suppl 1): S4-S13.

[50] CirilloS, UrenaJF, LambertJD, et al.. Impact of electronic cigarette heating coil resistance on the production of reactive carbonyls, reactive oxygen species and induction of cytotoxicity in human lung cancer cells in vitro. Regul Toxicol Pharmacol., 2019, 109. 104500

[51] LuoZ, XuX, ShoT, et al.. ROS-induced autophagy regulates porcine trophectoderm cell apoptosis, proliferation, and differentiation. Am J Physiol Cell Physiol., 2019, 316(2): C198-C209.

[52] XiaY, BrewerA, BellJT. DNA methylation signatures of incident coronary heart disease: findings from epigenome-wide association studies. Clin Epigenetics., 2021, 131186.

[53] CaoJ, WuQ, HuangY, et al.. The role of DNA methylation in syndromic and non-syndromic congenital heart disease. Clin Epigenetics., 2021, 13193.

[54] BisserierM, MathiyalaganP, ZhangS, et al.. Regulation of the methylation and expression levels of the BMPR2 gene by SIN3a as a novel therapeutic mechanism in pulmonary arterial hypertension. Circulation., 2021, 144(1): 52-73.

[55] LiH, JiB, XuT, et al.. Antenatal hypoxia affects pulmonary artery contractile functions via downregulating L-type Ca(2+) channels subunit Alpha1 C in adult male offspring. J Am Heart Assoc., 2021, 108. e019922

[56] XuC, JiangS, MaX, et al.. CRISPR-based DNA methylation editing of NNT rescues the cisplatin resistance of lung cancer cells by reducing autophagy. Arch Toxicol., 2023, 97(2): 441-456.

[57] TingL, FengY, ZhouY, et al.. IL-27 induces autophagy through regulation of the DNMT1/lncRNA MEG3/ERK/p38 axis to reduce pulmonary fibrosis. Respir Res., 2023, 24167.

[58] TaoH, ShiP, XuanHY, et al.. DNA methyltransferase-1 inactivation of androgen receptor axis triggers homocysteine induced cardiac fibroblast autophagy in diabetic cardiac fibrosis. Arch Biochem Biophys., 2020, 692. 108521

[59] GaoX, GaoX, ZhangY, et al.. Associations of self-reported smoking, cotinine levels and epigenetic smoking indicators with oxidative stress among older adults: a population-based study. Eur J Epidemiol., 2017, 32(5): 443-456.

[60] YaraS, LavoieJC, LevyE. Oxidative stress and DNA methylation regulation in the metabolic syndrome. Epigenomics., 2015, 7(2): 283-300.

[61] De PlanoLM, SaittaA, OddoS, et al.. Epigenetic changes in Alzheimer’s disease: DNA methylation and histone modification. Cells., 2024, 138719.

[62] ZhaoY, FanX, WangQ, et al.. ROS promote hyper-methylation of NDRG2 promoters in a DNMTS-dependent manner: Contributes to the progression of renal fibrosis. Redox Biol., 2023, 62. 102674

[63] DuraisamyAJ, MishraM, KowluruA, et al.. Epigenetics and regulation of oxidative stress in diabetic retinopathy. Invest Ophthalmol Vis Sci., 2018, 59(12): 4831-4840.

[64] WuQ, NiX. ROS-mediated DNA methylation pattern alterations in carcinogenesis. Curr Drug Targets., 2015, 16(1): 13-19.

[65] UysalF, SukurG, BozdemirN, et al.. Antioxidant supplementation may effect DNA methylation patterns, apoptosis, and ROS levels in developing mouse embryos. Histochem Cell Biol., 2024, 162(3): 215-224.

[66] FengX, ZhangH, MengL, et al.. Hypoxia-induced acetylation of PAK1 enhances autophagy and promotes brain tumorigenesis via phosphorylating ATG5. Autophagy., 2021, 17(3): 723-742.

[67] LiY, ZhangY, WalayatA, et al.. The regulatory role of H19/miR-181a/ATG5 signaling in perinatal nicotine exposure-induced development of neonatal brain hypoxic-ischemic sensitive phenotype. Int J Mol Sci., 2022, 23136885.

[68] JianJ, ZhangP, LiY, et al.. Reprogramming of miR-181a/DNA methylation patterns contribute to the maternal nicotine exposure-induced fetal programming of cardiac ischemia-sensitive phenotype in postnatal life. Theranostics., 2020, 10(25): 11820-11836.

Funding

National Natural Science Foundation of China(82300268)

Guangzhou Municipal Science and Technology Project(2023B03J1255)

RIGHTS & PERMISSIONS

The Author(s)