Z47 - The Superimposition of Cigarette Smoking and Menopause: How Chronic Exposure and Reproductive Senescence Intersect in Aortic Function

Introduction: : Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality worldwide, as the elderly population continues to grow. CVD incidence is significantly higher in males then females through middle age, but during the menopausal transition the cardioprotective effects of estrogen decline, and post-menopause CVD risk factors like hypertension develop faster in females than age-matched males. Yet, despite this clear mechanistic difference, preclinical CVD research in animal models remains overwhelmingly biased, with over 70% of studies solely including male mice. Furthermore, less than 1% of CVD preclinical studies utilize a human-like menopausal phenotype [1]. Accounting for menopause is crucial when evaluating the cardiovascular impact of environmental stressors that encompass a significant portion of the lifespan. Among these environmental stressors and other modifiable characteristics, combustible cigarette smoking is the single greatest risk factor for myocardial infarction (MI), one of the most severe complications from coronary artery disease (CAD) [2]. Despite ordinarily being more protected from CVD, female smokers are 25% more likely to develop CAD and have higher mortality rate than their male counterparts [3]. As of 2022, women 45-64 years of age have the highest rate of daily or near daily cigarette usage in the US at 14.8% compared to other age groups [4].

Materials and

Methods: : To address these limitations, we combined a preclinical model of menopause with cigarette smoke exposure, to determine the superimposed effect of these stressors on cardiovascular health. We replicated the human transition from perimenopause to menopause in female C57BL/6 mice by administering 4-vinylcyclohexene diepoxide (VCD), a selectively ovotoxic molecule that induces follicular atresia while leaving the ovaries intact, by imposing a gradual decline and cessation of non-androgenic endocrine production [5] . A 20-day course of VCD at 160mg/kg/ day resulted in menopausal transition by day 85. Following confirmed persistent diestrus, mice were exposed to cigarette smoke or air for 1 hour/day, 5 days/week, for 20 weeks. Our study thus included 4 groups, labelled as: VCD-Cig, VCD-Air, Estrous-Cig, and Estrous-Air (see Fig. 1). Functional changes to the descending thoracic aorta (DTA) were quantified via vasoactive biaxial testing using phenylephrine (Phen) for vasoconstriction and acetylcholine (Ach) for vasodilation. Vasodilation is largely mediated by the production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS). Diffusion of NO from endothelial cells (EC ) to vascular smooth muscle cells (VSMC) triggers relaxation. A second Ach challenge with the eNOS inhibitor L-NAME was used to determine the contribution of eNOS to EDD. The difference in Ach-induced vasodilation without and with L-NAME is a proxy for NO bioavailability. We hypothesized that, due to loss of circulating estradiol among other hormonal changes, VCD-treated mice would exhibit impaired aortic vasodilation, and that the effect would be further amplified by daily exposure to cigarette smoke.

Results, Conclusions, and Discussions:: The menopausal vascular phenotype exhibited two main features, increased contractility and impaired vasodilation in response to adrenergic and cholinergic stimuli, respectively, both of which were exacerbated by cigarette smoking. VCD-Cig aortas were the most contractile (18.0%), followed by VCD-Air (13.0%), Estrous-Cig (8.1%), and Estrous-Air (6.1%) specimens (Fig. 2e). Ach administration resulted in limited endothelium-dependent vasodilation (EDD) for VCD-Cig vessels (57% recovery), and specimens from the VCD-Air mice showed greater EDD impairment than specimens from the Estrous-Cig group (65% and 72% recovery, respectively, Fig. 2 a-f). As expected, aortas in the Estrous-Air groups recovered to 98% of the original diameter. In the L-NAME test, aortas from Estrous-Air mice recovered by 59.3%, Estrous-Cig by 50.4%, VCD-Air by 60.7%, and VCD-Cig by 46.0% (Table 3). These results align with clinical findings of hypertension and increased CVD risk following menopause and in female smokers. It has been shown that oxidative stress induced by cigarette smoking reduces NO availability by uncoupling eNOS, downregulating eNOS, and scavenging NO. On the other hand, estradiol has antioxidant and pro-dilatory properties, upregulates eNOS, and triggers non-canonical NO production. Therefore, postmenopausal female mice may no longer have sufficient circulating estradiol to inhibit the pathologic effects of smoking.

Overall, our work confirms that the menopausal transition presents a turning point for endothelial function and further suggests that the menopausal endocrine shift primes the vasculature for dysfunction when combined with other sources of oxidative stress. Endothelial dysfunction is the primum movens of CVD, triggering a cascade of events resulting in hypertension, arterial stiffening, and heart disease. The strong association between menopause and endothelial dysfunction, supports the “healthy endothelium hypothesis” which advocates for addressing hormonal changes early during the menopausal transition to reduce CVD risk by preventing endothelial dysfunction.

Ultimately, accounting for menopausal transition and menopause is necessary for translational relevance when studying aging-related disease in preclinical models. By excluding these effects, we may overlook the specific risks that over half of the aging population faces . Superposing menopause with persistent environmental stressors in preclinical models is thus imperative for advancing our understanding of the mechanisms that underly sex-dependent disease and risk disparities.

Acknowledgements and/or References (Optional):: ACKNOWLEDGEMENTS: We acknowledge financial support from the NIH, award R01HL168719. Additionally, thank you to lab members Simone Sabnis and Ana Vargas for their assistance in dissections for active testing and Grace Kelly for assisting in exposures and monitoring.

REFERENCES
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