Long-Term Adverse Effects of Perinatal Hypoxia on the Adult Pulmonary Circulation Vary Between Males and Females in a Murine Model

Abstract

Adverse events during the perinatal period are associated with an increased risk to develop cardiometabolic diseases later in life. We established a murine model to study long-term effects of perinatal hypoxia (PH) on the pulmonary circulation. We previously demonstrated that PH led to an impaired regulation of pulmonary vascular tone in adulthood, linked to alterations in K+ channels in males and in the nitric oxide (NO)/cyclic guanosine monophosphate pathway in females. Moreover, simultaneous administration of inhaled NO (iNO) during PH exposure prevented adverse effects of PH on adult pulmonary vasculature in females. The present study showed that PH induced a significant increase in right ventricular pressure in males and females, and an enhanced sensitivity to acute hypoxia in females. PH significantly reduced acetylcholine-induced relaxation in pulmonary artery, to a greater extent in females than in males. PH led to right ventricular hypertrophy in adulthood, appearing earlier in males than in females. Morphometric measurements showed a significant increase in the number of 25-75-µm pulmonary vessels in male lungs following PH, probably resulting in increased pulmonary vascular resistance. The effects of prolonged hypoxia in adulthood differed between males and females. Perinatal iNO during PH prevented PH-induced alterations in the cardiopulmonary system, whereas perinatal iNO alone could have some adverse effects. Therefore, PH led to long-lasting alterations in the regulation of adult pulmonary circulation, which vary between males and females. In males, the increased pulmonary vascular resistance was associated with morphological changes besides functional alterations, whereas females showed an important pulmonary vascular dysfunction. Keywords: Perinatal hypoxia, Pulmonary circulation, Endothelium-dependent relaxation, Phosphodiesterases, Sex differences.

Fig. 1

Systolic right ventricular pressure (RVP) measured under normoxia (21 % O₂) or acute hypoxia (12 % O₂) in males (M) and females (F). Closed-chest measurements were performed in adult mice born in normoxia (Ctr) or hypoxia (PH), with or without a 5-day exposure to chronic hypoxia in adulthood (AH). Graphs A–C present individual values with bar at mean ± SEM. (A) RVP in Ctr and PH males and females (n=4–8); (B) RVP in Ctr and PH males with or without AH (n=3–11); data were analyzed by two-way ANOVA (results are shown below) with Sidak’s multiple comparison test (significant p values are reported on the graph). (C) RVP increase between normoxia and acute hypoxia, expressed as percentage of RVP in normoxia (n=3–6); data were analyzed by one-way ANOVA and unpaired t tests with Welch’s correction to compare two groups (significant p values are reported on the graph). (D) RVP measured in males during the transition from normoxia to acute hypoxia, followed by addition of increasing concentrations of inhaled nitric oxide (iNO) in the 12 % O₂ gas mixture; data are expressed as mean ± SEM (n=3–11) and were analyzed by two-way ANOVA (results are shown below) with Sidak’s multiple comparison test; significant difference (p<0.05): * PH or Ctr+AH versus Ctr; † PH+AH or Ctr+AH versus PH; ‡ PH+AH versus Ctr+AH.

Fig. 2

Relaxation induced by cumulative doses of acetylcholine (ACh) or 8-bromo-cGMP in pulmonary arteries of males (M) and females (F) born in normoxia (Ctr) or hypoxia (PH), with or without a 5-day exposure to chronic hypoxia in adulthood (AH), or with perinatal exposure to inhaled nitric oxide (NO). Vascular rings were pre-constricted with phenylephrine 10⁻⁵M. Data are expressed as mean ± SEM of the percentage of change in tension induced by the vasodilator (A n=6–8; B n=7–16; C n=5–9; D n=7–9; E n=6–14; F n=7–16). Data were analyzed by two-way ANOVA (results are shown below each graph) with Sidak’s multiple comparison test; significant difference (p<0.05): * PH versus Ctr; # Ctr+AH vs Ctr; † PH+AH or Ctr+AH or PH+NO versus PH; ‡ PH+AH versus Ctr+AH or PH+NO versus Ctr+NO. The corresponding AUC were presented next to each graph; data are expressed as mean ± SEM and were analyzed by unpaired t tests with Welch’s correction to compare two groups (significant p values are reported on each graph) (G). Schematic representation of the NO/cGMP relaxing pathway and interactions with pharmacological agents. eNOS, endothelial nitric oxide synthase; M3AChR, muscarinic acetylcholine receptor M3; PDEs, phosphodiesterases; PKG, cGMP-dependent protein kinase; sGC, soluble guanylyl cyclase. Dose-response curves to ACh assessed in Ctr and PH females were previously published in Peyter et al. [20]. Dose-response curves to ACh assessed in Ctr, PH and PH+NO females were previously published in Peyter et al. [21].

Fig. 3

Relaxation induced by cumulative doses of acetylcholine (ACh) in pulmonary arteries of females (F) born in normoxia (Ctr) or hypoxia (PH). Vascular rings were pre-incubated with phosphodiesterase (PDE) inhibitors (A, IBMX; C, 8-MM-IBMX; E, sildenafil; G, milrinone) and pre-constricted with phenylephrine 10⁻⁵M. Some mice were treated with sildenafil during 5 days before testing the reactivity of their pulmonary arteries in the absence of PDE inhibitor (I). Data are expressed as mean ± SEM of the percentage of change in tension induced by ACh (A n=6–8; C n=6–7; E n=6–11; G n=6–11; I n=6–8). Data were analyzed by two-way ANOVA (results are shown below each graph) with Sidak’s multiple comparison test; significant difference (p<0.05): * PH versus Ctr; † PH+PDE inhibitor versus PH. The corresponding AUC were presented next to each graph; data are expressed as mean ± SEM and were analyzed by unpaired t tests with Welch’s correction to compare two groups (significant p values are reported on each graph). Dose-response curves to the PDE inhibitors (B, IBMX; D, 8-MM-IBMX; F, sildenafil; H, milrinone) were established in pulmonary arteries pre-constricted with phenylephrine 10⁻⁵M. Data are expressed as mean ± SEM of the percentage of change in tension induced by the PDE inhibitor (B n=8; D n=5–11; F n=8; H n=7–8). Data were analyzed by two-way ANOVA with Sidak’s multiple comparison test; * significant difference between PH and Ctr mice. The corresponding AUC were presented next to each graph; data are expressed as mean ± SEM and were analyzed by unpaired t tests with Welch’s correction. Dose-response curves to ACh assessed in Ctr and PH females were previously published in Peyter et al. [20].

Fig. 4

Cardiopulmonary parameters at rest and after a 30-min swimming challenge in females born in normoxia (Ctr) or hypoxia (PH), with or without perinatal exposure to inhaled nitric oxide (NO). Oxygen saturation (sat O2) (A, D, G), pulse rate (B, E, H) and breath rate (C, F, I) were recorded in anesthetized females at rest (A–C), conscious females at rest (D–F) and females anesthetized after 30-min swimming in normoxic or hypoxic conditions (G–I). For females at rest, cardiopulmonary data were recorded during the transition from normoxia (21 % O₂) to acute hypoxia (12 % O₂); for females submitted to exercise, the recordings were performed in the same condition (normoxia or hypoxia) as the swimming challenge. Individual values are presented with bar at mean ± SEM (A–C n=7–10; D–F n=7–10; G–I n=9–10). Data were analyzed by two-way ANOVA (results are shown below each graph) with Sidak’s multiple comparison test (significant p values are reported on the graph).

Fig. 5

Morphometric analysis of adult lungs from males (M) born in normoxia (Ctr) or hypoxia (PH), with or without a 5-day exposure to chronic hypoxia in adulthood (AH). Individual values are presented with bar at mean ± SEM (n=3–4). (A) The counted vessels were categorized by size; data are expressed as number of vessels per area analyzed; data were analyzed by two-way ANOVA (results are shown below) with Sidak’s multiple comparison test (significant p values are reported on the graph). (B) Total number of vessels per area analyzed; data were analyzed by two-way ANOVA followed by Sidak’s multiple comparison test.