Ovarian hormones do not mediate protection against pulmonary hypertension and right ventricular remodeling in female mice exposed to chronic, inhaled nicotine

Abstract

Electronic cigarette use has increased globally prompting calls for improved understanding of nicotine’s cardiovascular health effects. Our group has previously demonstrated that chronic, inhaled nicotine induces pulmonary hypertension and right ventricular (RV) remodeling in male mice, but not female mice, suggesting sex differences in nicotine-related pathology. Clinically, biological females develop pulmonary hypertension more often but have less severe disease than biological males, likely because of the cardiopulmonary protective effects of estrogen. Nicotine is also metabolized more rapidly in biological females because of differences in cytochrome-P450 activity, which are thought to be mediated by female sex hormones. These findings led us to hypothesize that female mice are protected against nicotine-induced pulmonary hypertension by an ovarian hormone-dependent mechanism. In this study, intact and ovariectomized (OVX) female mice were exposed to chronic, inhaled nicotine or room air for 12 h/day for 10–12 wk. We report no differences in serum cotinine levels between intact and OVX mice. In addition, we found no structural (RV or left ventricular dimensions and Fulton index) or functional (RV systolic pressure, pulmonary vascular resistance, cardiac output, ejection fraction, and fractional shortening) evidence of cardiopulmonary dysfunction in intact or OVX mice. We conclude that ovarian hormones do not mediate cardiopulmonary protection against nicotine-induced pulmonary hypertension. Due to profound sex differences in clinical pulmonary hypertension pathogenesis and nicotine metabolism, further studies are necessary to elucidate mechanisms underlying protection from nicotine-induced pathology in female mice.

NEW & NOTEWORTHY The emergence of electronic cigarettes poses a threat to cardiovascular and pulmonary health, but the direct contribution of nicotine to these disease processes is largely unknown. Our laboratory has previously shown that chronic, inhaled nicotine induces pulmonary hypertension and right ventricular remodeling in male mice, but not female mice. This study using a bilateral ovariectomy model suggests that the cardiopulmonary protection observed in nicotine-exposed female mice may be independent of ovarian hormones.

INTRODUCTION

Worldwide trends in electronic cigarette (e-cig) use have prompted leaders from the American Heart Association, World Heart Federation, American College of Cardiology, and European Society of Cardiology to issue a joint opinion urging increased efforts toward ending the tobacco epidemic (1). Analysis of the National Health and Nutrition Examination Surveys (2013–2018) led to the inclusion of e-cig vaping in 2022’s “Life’s Essential 8” measurement of cardiovascular risk factors published by the American Heart Association (2). Although recent population surveys indicate declining rates of current e-cig use in American adults, the rates of daily e-cig use have increased among users, raising concerns about rising nicotine dependence (3). Cigarette smoking has been linked to countless cardiovascular and pulmonary diseases, but the direct contributions of nicotine to these processes are unclear (4). The emergence of e-cigs (and other novel tobacco products) has elicited calls for further research toward understanding the health implications of nicotine exposure (1).

Pulmonary hypertension (PH), characterized by elevated blood pressure in the pulmonary circulation and the right heart, has been linked to cigarette smoking in humans and rodents (5–7). Male mice exposed to inhaled, heated e-cig liquid containing 24 mg/mL of nicotine for 6 mo developed cardiopulmonary changes consistent with PH: increased right ventricular (RV) free wall thickness during systole and diastole (RV FWT;s and; d), RV dysfunction, and inflammation of the RV and lungs (8). Our laboratory has previously demonstrated that chronic, inhaled nicotine leads to PH and RV remodeling in male mice associated with alterations in the renin-angiotensin system and in nitric oxide signaling (9–11). Male mice exposed to inhaled nicotine for up to 12 wk demonstrate increased RV FWT;d, increased RV internal diameter during diastole (RVID;d), and elevated RV systolic pressure (RVSP; 10, 11). In contrast, female mice exposed to the same conditions are protected against these PH-associated changes (9).

PH exhibits profound sex differences. Clinically, biological females develop PH more often but have less severe disease than biological males, likely because of cardiopulmonary protective effects of estrogen (12). Biological females with PH have reduced RV remodeling and improved RV function versus biological males (13). This RV-protective phenotype has been recapitulated in female rodents using a Sugen/hypoxia model of PH (14, 15). Sex differences in patients with PH are reduced with increased age, suggesting that menopausal changes in ovarian hormones play an important role in PH pathogenesis (13, 16). In our studies, serum cotinine levels (a marker of nicotine exposure) are lower in female mice than male mice despite identical exposure paradigms (9, 17). Consistent with our findings in mice, biological female patients metabolize nicotine more rapidly than biological males, likely because of estrogen-mediated differences in cytochrome-P450 activity (18). Biological females taking estrogen-containing contraceptives metabolize nicotine more rapidly than biological females taking progesterone-only contraceptives or no contraceptives (18). These findings, taken together, led us to hypothesize that female mice are protected against nicotine-induced PH and RV remodeling by an ovarian hormone-dependent mechanism.

MATERIALS AND METHODS

Animals

Adult, female C57BL6/J mice were purchased from Jackson Laboratory (Bar Harbor, ME). Cages were randomized to intact-air, ovariectomy (OVX)-air, intact-nicotine, or OVX-nicotine groups. Mice (10 wk old) in the OVX groups underwent bilateral OVX 1 wk before nicotine exposure. Mice were housed in a temperature- and humidity-controlled (19°C–25°C and 45–60% humidity) facility under a 12-h:12-h dark/light cycle. Mice received standard chow (iOS Teklab Extruded Rodent Diet 2019S; Envigo, Huntingdon, UK) and water ad libitum. All procedures conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Louisiana State University Health Sciences Center Institutional Animal Care and Use Committee (Protocol No. 3674).

Chronic Nicotine Inhalation Model

Mice were housed in a nicotine inhalation chamber (La Jolla Alcohol Research, La Jolla, CA) as previously described (9–11). Briefly, unheated nicotine vapor was produced by bubbling room air into a pure nicotine solution (≥99% free base liquid; Sigma-Aldrich, St. Louis, MO). The nicotine vapor concentrate was diluted with additional room air and distributed between chambers. Air flow was adjusted to achieve serum cotinine levels comparable to human electronic or combustible cigarette users (19, 20). Nicotine exposure occurred during a 12-h-on/12-h-off schedule that overlapped the dark cycle. Intact and OVX mice in the air-exposure group were housed in the same room, but outside of the inhalation chamber. Blood samples were collected biweekly using submandibular vein puncture at the conclusion of a nicotine-exposure period; serum cotinine was assessed by ELISA in these postexposure samples (Calbiotech, El Cajon, CA).

Echocardiography

B-mode and M-mode echocardiographic images of the RV and left ventricle (LV) were recorded using the Vevo 3100 Imaging System with a 30-MHz probe (VisualSonics, Toronto, Canada) at baseline and 8 wk of nicotine exposure. Echocardiography was performed under 1–1.5% isoflurane anesthesia with mice placed on a heated pad, maintaining heart rates between 400 and 550 beats/min. Imaging processing used the leading-edge method in the Vevo LAB software (VisualSonics) during a minimum of three cardiac cycles.

RV Pressure Measurement

Following 10–12 wk of exposure, mice underwent catheterization for measurement of RVSP. Mice were anesthetized with 2–3% isoflurane and placed on a heated pad. A pressure transducer (SPR-1000; Millar, Houston, TX) was inserted into the RV through the right jugular vein and right atrium. RV pressure was recorded and analyzed using the PowerLab 8/35 system (ADInstruments, Colorado Springs, CO).

Plasma and Tissue Collection

At the conclusion of the study, plasma was isolated from venous blood collected from the jugular vein into lithium heparin-coated microtubes (BD Biosciences, Franklin Lakes, NJ). Mice under deep anesthesia were euthanized by exsanguination. The heart was collected. The RV was separated from LV and interventricular septum (LV + S), and tissues were weighed individually. Tibial length was measured. Plasma estrogen was assessed using a mouse estrogen ELISA kit (ab285291, Abcam, Cambridge, UK).

Statistical Analysis

Data are expressed as means ± SE. One mouse died during OVX and was excluded from all measurements; another died during catheterization and was excluded from RVSP and pulmonary vascular resistance (PVR) measurements. Findings were analyzed by two-way ANOVA or F test of simple linear regression using GraphPad Prism 9 (GraphPad Software, San Diego, CA). P < 0.05 was considered statistically significant.

RESULTS

Characterization of Nicotine Exposure and OVX

We have previously shown a PH phenotype in male, but not female mice, exposed to chronic, inhaled nicotine (9). This study aimed to examine the effects of ovarian hormones on protection against nicotine-induced pathology using bilateral OVX. Total plasma estrogen was not significantly different between intact-air mice (499 ± 60 pg/mL, n = 5) and intact-nicotine mice (525 ± 37 pg/mL, n = 5). There was a significant reduction in total plasma estrogen in OVX mice (Fig. 1A, P < 0.01). There were not, however, significant differences in plasma estrogen between OVX-air mice (336 ± 44 pg/mL, n = 5) and OVX-nicotine mice (421 ± 31 pg/mL, n = 5). Estrogen was comparable between OVX females and age-matched males (Supplemental Fig. S1; see https://doi.org/10.6084/m9.figshare.21225425.v1). Serum cotinine, a nicotine metabolite with extended half-life, was significantly elevated in mice exposed to chronic, inhaled nicotine versus air-exposed mice (Fig. 1B, P < 0.0001). Although previous studies in humans and mice report estrogen-mediated changes in nicotine metabolism (18, 21), we found no differences in serum cotinine between intact-nicotine mice (421 ± 101 ng/mL, n = 9) and OVX-nicotine mice (314 ± 61 ng/mL, n = 8). There was a main effect of nicotine to reduce the percent body weight change (Fig. 1C, P < 0.0001). Consistent with other studies of mouse OVX, we report a main effect of OVX to increase percent body weight change (P < 0.05; 22).

Figure 1.

Study characteristics of female mice following nicotine exposure and ovariectomy (OVX). A: nicotine exposure does not affect OVX-induced reductions in plasma estrogen measured by ELISA, n = 5/group. B: OVX does not affect group-averaged serum cotinine levels measured by ELISA in air- or nicotine-exposed mice, n = 8–10/group. C: body weight is reduced by nicotine exposure and increased by OVX, n = 8–10/group. D and E: left ventricular plus septal (LV + S) and right ventricular (RV) weights indexed to tibial length are reduced by nicotine exposure and are unaffected by OVX; n = 8 and 10/group. F: Fulton index, a marker of RV hypertrophy, is unaffected by both nicotine exposure and OVX; n = 8–10/group. G: Fulton index is not correlated with estrogen levels in intact (n = 5) or OVX (n = 5) mice exposed to nicotine. Data are displayed as means ± SE. *P < 0.05, **P < 0.01, and ****P < 0.0001 are main effects of nicotine exposure; #P < 0.05 and ##P < 0.01 are main effects of OVX. These data are analyzed using two-way ANOVA (A–F) or F test of simple linear regression (G).

Chronic, Inhaled Nicotine Reduces RV and LV Weight, but Not Fulton Index, in an Ovarian Hormone-Independent Manner

There was a main effect of nicotine to reduce both RV weight indexed to tibial length (Fig. 1D, P < 0.05) and LV + S weight indexed to tibial length (Fig. 1E, P < 0.01). These nicotine-driven changes in cardiac tissue weight are unaffected by OVX. Fulton index is a marker of RV hypertrophy, which is calculated by dividing RV weight by LV + S weight. We found no differences in Fulton index between intact-air mice (25.3 ± 0.68, n = 8), intact-nicotine mice (24.2 ± 0.76, n = 10), OVX-air mice (27.3 ± 1.47, n = 9), and OVX-nicotine mice (25.8 ± 0.64, n = 10, Fig. 1F). Furthermore, there was no correlation between Fulton index and plasma estrogen in intact and OVX mice exposed to nicotine (Fig. 1G, r = −0.25, P = 0.48).

Protection from RV Hypertrophy in Chronic Nicotine Inhalation is Not Affected by OVX

RV remodeling is an important indicator of poor prognosis in PH (23). In contrast to our previous findings in male mice, chronic, inhaled nicotine does not induce RV remodeling in intact or OVX female mice (9–11). We found no significant differences in RV free wall thickness during diastole (RV FWT;d) between intact-air mice (0.33 ± 0.02 mm, n = 8), intact-nicotine mice (0.29 ± 0.02 mm, n = 10), OVX-air mice (0.32 ± 0.02 mm, n = 8), and OVX-nicotine mice (0.29 ± 0.01 mm, n = 10, Fig. 2, A and B). There was no correlation between RV FWT;d and plasma estrogen in intact and OVX mice exposed to nicotine (Fig. 2C, r = 0.05, P = 0.89). We also found no significant differences in RV internal diameter during diastole (RVID;d) between intact-air mice (1.30 ± 0.03 mm, n = 8), intact-nicotine mice (1.34 ± 0.04 mm, n = 10), OVX-air mice (1.23 ± 0.05 mm, n = 8), and OVX-nicotine mice (1.24 ± 0.05, n = 10, Fig. 2, A and D). There was no correlation between RVID;d and plasma estrogen in intact and OVX mice exposed to nicotine (Fig. 2E, r = 0.49, P = 0.15).

Figure 2.

Neither nicotine exposure nor ovariectomy (OVX) affect echocardiographic measurements of right ventricular (RV) remodeling. A: representative B-Mode and M-Mode echocardiographic images of the RV. B: RV free wall thickness during diastole (RV FWT;d) is unaffected by nicotine exposure and OVX; n = 8–10/group. C: RV FWT;d is not correlated with estrogen levels in intact (n = 5) or OVX (n = 5) mice exposed to nicotine. D: RV internal diameter during diastole (RVID;d) is unaffected by nicotine exposure and OVX; n = 8–10/group. E: RVID;d is not correlated with estrogen levels in intact (n = 5) or OVX (n = 5) mice exposed to nicotine. Data are displayed as means ± SE. These data are analyzed using two-way ANOVA (B and D) or F test of simple linear regression (C and E). AO, aorta; LV, left ventricle; PA, pulmonary artery.

Protection from Cardiopulmonary Dysfunction in Chronic Nicotine Inhalation is Not Affected by OVX

In contrast to our previous findings in male mice, chronic, inhaled nicotine does not induce cardiopulmonary dysfunction in intact or OVX female mice (9–11). We found no significant differences in RV systolic pressure (RVSP) between intact-air mice (24.0 ± 0.48 mmHg, n = 7), intact-nicotine mice (22.7 ± 0.44 mmHg, n = 9), OVX-air mice (24.4 ± 0.88 mmHg, n = 9), and OVX-nicotine mice (23.6 ± 0.56 mmHg, n = 10, Fig. 3A). There was no correlation between RVSP and plasma estrogen in intact and OVX mice exposed to nicotine (Fig. 3B, r = −0.19, P = 0.62). We found no significant differences in cardiac output (CO) between intact-air mice (15.5 ± 1.2 mL/min, n = 8), intact-nicotine mice (15.0 ± 0.4 mL/min, n = 10), OVX-air mice (16.5 ± 0.7 mL/min, n = 8), and OVX-nicotine mice (16.8 ± 0.5 mL/min, n = 10, Fig. 3C). There was no correlation between CO and plasma estrogen in intact and OVX mice exposed to nicotine (Fig. 3D, r = −0.38, P = 0.27). We found no significant differences in PVR between intact-air mice (1.56 ± 0.16 mmHg·min·mL⁻¹, n = 7), intact-nicotine mice (1.50 ± 0.07 mmHg·min·mL⁻¹, n = 9), OVX-air mice (1.51 ± 0.11 mmHg·min·mL⁻¹, n = 8), and OVX-nicotine mice (1.43 ± 0.05 mmHg·min·mL⁻¹, n = 10, Fig. 3E). There was no correlation between PVR and plasma estrogen in intact and OVX mice exposed to nicotine (Fig. 3F, r = 0.35, P = 0.36).

Figure 3.

Neither nicotine exposure nor ovariectomy (OVX) affect cardiopulmonary function. A: RV systolic pressure (RVSP) is unaffected by nicotine exposure and OVX; n = 7–10/group. B: RVSP is not correlated with estrogen levels in intact (n = 4) or OVX (n = 5) mice exposed to nicotine. C: cardiac output (CO) is unaffected by nicotine exposure and OVX; n = 8–10/group. D: CO is not correlated with estrogen levels in intact (n = 5) or OVX (n = 5) mice exposed to nicotine. E: pulmonary vascular resistance (PVR) is unaffected by nicotine exposure and OVX; n = 7–10/group. F: PVR is not correlated with estrogen levels in intact (n = 4) or OVX (n = 5) mice exposed to nicotine. Data are displayed as means ± SE. These data are analyzed using two-way ANOVA (A, C, and E) or F test of simple linear regression (B, D, and F).

There was a main effect of nicotine (P < 0.05) to decrease LV internal diameter during systole (LVID;s) in intact (2.55 ± 0.05 mm, n = 10) and OVX (2.58 ± 0.06 mm, n = 10) nicotine-exposed mice versus air-exposed mice (intact: 2.76 ± 0.07 mm, n = 8; OVX: 2.68 ± 0.09 mm, n = 8). We found no additional differences in LV chamber dimensions, heart rate, ejection fraction, or fractional shortening with nicotine exposure or OVX (Table 1).

Table 1.

Left ventricular echocardiographic measurements

	Intact		Ovx
Echocardiographic Measurement	Air	Nicotine	Air	Nicotine
n	8	10	8	10
Heart rate, beats/min	463 ± 12	443 ± 11	430 ± 11	456 ± 18
LVID;s, mm	2.76 ± 0.07	2.55 ± 0.05*	2.68 ± 0.09	2.58 ± 0.06*
LVID;d, mm	3.80 ± 0.05	3.68 ± 0.04	3.87 ± 0.07	3.78 ± 0.06
LVPW;s, mm	1.02 ± 0.02	1.06 ± 0.04	1.01 ± 0.02	1.03 ± 0.04
LVPW;d, mm	0.79 ± 0.02	0.77 ± 0.04	0.72 ± 0.03	0.72 ± 0.02
EF, %	53.7 ± 2.52	59.3 ± 1.43	59.3 ± 1.77	60.7 ± 1.14
FS, %	27.4 ± 1.70	30.9 ± 0.96	31.0 ± 1.19	31.9 ± 0.78

Values are means ± SE; n, number of subjects. EF, ejection fraction; FS, fractional shortening; LV, left ventricle; LVID;s and LVID;d, LV internal diameter during systole and diastole, respectively; LVPW;s and LVPW;d, LV posterior wall thickness during systole and diastole, respectively; OVX, ovariectomy. Two-way ANOVA. *P < 0.05 is a main effect of nicotine exposure.

DISCUSSION

Previous studies have shown PH and RV remodeling in male mice exposed to chronic, inhaled nicotine and nicotine-containing e-cigs (8, 10, 11). In contrast, female mice exposed to chronic, inhaled nicotine do not develop PH or RV remodeling (9). In this study, we aimed to assess the role of ovarian hormones in cardiopulmonary protection against nicotine-induced pathology using bilateral OVX. Despite reduced estrogen levels in OVX female mice versus intact female mice, we found no differences in serum cotinine, Fulton index, RV FWT;d, RVID;d, RVSP, CO, or PVR between intact and OVX females exposed to chronic, inhaled nicotine. In addition, we report no correlation between plasma estrogen levels and these parameters in nicotine-exposed female mice. This led us to conclude that ovarian hormones do not mediate cardiopulmonary protection against nicotine-induced PH.

PH is a disease state with profound sex differences in humans, with greater susceptibility in biological females versus biological males (12, 13). Although biological females develop PH more often than biological males, biological females have reduced incidence of maladaptive RV remodeling and have improved prognosis (13). Many of the sex differences in PH pathogenesis are reduced with aging, suggesting a protective role of ovarian hormones (13, 16). In addition to estrogen, other hormones including testosterone, progesterone, and dehydroepiandrosterone have been implicated in PH sex differences (13). Cigarette smoking upregulates these hormones in postmenopausal women (24). Sex hormone-independent mechanisms including sexual dimorphism in the immune system, iron homeostasis, and bone morphogenetic protein receptor type 2 expression have also been implicated (13).

Estrogen-dependent upregulation of cytochrome-P450 expression and activity is associated with accelerated nicotine metabolism in humans (18). This is consistent with our previous findings showing reduced serum cotinine levels in female mice versus male mice (9, 17). Others have also reported lower serum cotinine levels in female mice, which are accompanied by increased cytochrome-P450 mRNA expression and protection against LV dysfunction in comparison with male mice (21). Despite these estrogen-driven changes in nicotine metabolism, we found no significant difference in serum cotinine levels between intact nicotine-exposed mice and OVX nicotine-exposed mice. In addition, bilateral OVX reduced total plasma estrogens by ∼30%. As a result, our finding that nicotine metabolism was unaffected by bilateral OVX may be explained by extraovarian sources of estrogen (adipose tissue, liver, and others) or nonhormonal regulation of cytochrome-P450 (25, 26).

Future studies may require a modified nicotine exposure paradigm to achieve serum cotinine levels equivalent to those found in male mice. This study is limited by OVX in exclusively postpubertal mice. Although high estrogen doses during puberty may provide lasting cardioprotection after OVX, a human cohort study indicated that premature ovarian failure is an independent risk factor in the development of PH (27). Additional work could assess cardioprotection via OVX at various stages of development or via treatment of nicotine-exposed male mice with exogenous estrogen. A four core genotypes model (XX + ovaries, XX + testes, XY + ovaries, and XY + testes) may also provide insight into the role of androgens and hormone-independent sexual dimorphism in nicotine-induced disease (28).

Due to extensive sex differences in clinical PH pathogenesis and nicotine metabolism, further studies are necessary to elucidate mechanisms underlying cardiopulmonary protection against nicotine in female mice.

SUPPLEMENTAL DATA

Supplemental Fig S1: https://doi.org/10.6084/m9.figshare.21225425.v1.

GRANTS

This study was supported by National Heart, Lung, and Blood Institute Grants R01HL135635 and R01HL135635-S1 (to E.L, X.Y., J.D.G.) and F30HL160071 (to A.W.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

E.L., X.Y., and J.D.G. conceived and designed research; N.D.F., A.W., E.L., X.Y., and J.D.G. performed experiments; N.D.F., A.W., E.L., X.Y., and J.D.G. analyzed data; N.D.F., A.W., E.L., X.Y., and J.D.G. interpreted results of experiments; N.D.F. and A.W. prepared figures; N.D.F. drafted manuscript; N.D.F., A.W., E.L., X.Y., and J.D.G. edited and revised manuscript; N.D.F., A.W., E.L., X.Y., and J.D.G. approved final version of manuscript.