Neonatal hyperoxic lung injury favorably alters adult right ventricular remodeling response to chronic hypoxia exposure

Abstract

The development of pulmonary hypertension (PH) requires multiple pulmonary vascular insults, yet the role of early oxygen therapy as an initial pulmonary vascular insult remains poorly defined. Here, we employ a two-hit model of PH, utilizing postnatal hyperoxia followed by adult hypoxia exposure, to evaluate the role of early hyperoxic lung injury in the development of later PH. Sprague-Dawley pups were exposed to 90% oxygen during postnatal days 0-4 or 0-10 or to room air. All pups were then allowed to mature in room air. At 10 wk of age, a subset of rats from each group was exposed to 2 wk of hypoxia (Patm = 362 mmHg). Physiological, structural, and biochemical endpoints were assessed at 12 wk. Prolonged (10 days) postnatal hyperoxia was independently associated with elevated right ventricular (RV) systolic pressure, which worsened after hypoxia exposure later in life. These findings were only partially explained by decreases in lung microvascular density. Surprisingly, postnatal hyperoxia resulted in robust RV hypertrophy and more preserved RV function and exercise capacity following adult hypoxia compared with nonhyperoxic rats. Biochemically, RVs from animals exposed to postnatal hyperoxia and adult hypoxia demonstrated increased capillarization and a switch to a fetal gene pattern, suggesting an RV more adept to handle adult hypoxia following postnatal hyperoxia exposure. We concluded that, despite negative impacts on pulmonary artery pressures, postnatal hyperoxia exposure may render a more adaptive RV phenotype to tolerate late pulmonary vascular insults.

Keywords: atrial natriuretic peptide; capillarization; prematurity; pulmonary hypertension; right ventricular adaptation.

Fig. 1.

Timeline of experimental exposures. Brief hyperoxia (4-day, 4d) exposure is limited to the period of saccular lung development, whereas prolonged hyperoxia (10-day, 10d) exposure includes injury well into the bulk alveolarization phase. RA, room air; H, hypoxia; GA, gestational age.

Fig. 2.

Postnatal hyperoxia exposure leads to increased right ventricular systolic pressure (RVSP) and increased RV hypertrophy (RVH) after adult hypoxia exposure. Postnatal hyperoxia (Fi_O2 > 0.9) exposure occurred for the first 4 or 10 days of life, respectively. Hypoxia exposure (P_atm = 362 mmHg for 2 wk) occurred from week 10 to week 12. All end points were measured at 12 wk. A: pulmonary hypertension (PH) assessed by measurement of RVSP. PH develops after hypoxia exposure but is most severe in animals exposed to prolonged (10-day) postnatal hyperoxia. B: RVH assessment by Fulton index [weight of RV divided by weight of the left ventricle plus septum; RV/(LV + S)] following postnatal hyperoxia and adult hypoxia exposures. Note exaggerated RVH in animals exposed to postnatal hyperoxia and hypoxia. C: assessment of LV hypertrophy (reported as LV weight indexed to body weight) demonstrates similar LV hypertrophy among groups. D: mean arterial pressures (MAP) increase following hypoxia exposure but are not affected by hyperoxia exposure. Group labels are as outlined in Fig. 1. Points represent individual animals, and error bars represent means ± SE. Analysis by 1-way ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001 compared with RA control (RA-RA); #P < 0.05, ##P < 0.01, ###P < 0.001 compared with RA-hypoxia (RA-H).

Fig. 3.

Increased RVSP and increased RV hypertrophy in hyperoxia-hypoxia groups cannot be explained by increased pulmonary artery (PA) muscularization or decreased vascular density. A: Verhoff-van Gieson staining of lung demonstrates increased PA muscularization following hypoxia exposure with no additional effect of postnatal hyperoxia. PA muscularization was determined in a blinded fashion by dividing the measured pulmonary wall area by the entire vessel area. Representative images taken at ×20. Size bars = 50 μm. Note hypoxia-induced increase in PA wall thickness (indicated by arrowheads). B: von Willebrand factor staining demonstrates decreased pulmonary vascular density following hypoxia exposure (vessels marked by arrowheads). Note that animals exposed to prolonged neonatal hyperoxia had a small but statistically significant decrease in pulmonary microvessel density although pulmonary microvessel rarefaction was not exacerbated further by postnatal hypoxia exposure. Representative images taken at ×10. Size bars = 50 μm. Points represent individual animals, and error bars represent means ± SE. Analysis by 1-way ANOVA. *P < 0.05 compared with RA-RA.

Fig. 4.

Postnatal hyperoxia is associated with more preserved RV function and exercise capacity upon hypoxia exposure. A: cardiac output (CO) measured by echocardiography (ECHO) demonstrates cardiac dysfunction following hypoxia, with relative preservation of CO in animals exposed to prolonged postnatal hyperoxia. B: factional shortening, measured by the change in end-diastolic to end-systolic diameter over the end-diastolic diameter [(EDD − ESD)/EDD], was measured in a subset of hypoxia-exposed animals, again demonstrating significantly improved cardiac function in 10dO₂-H animals compared with RA-H. C: maximal exercise capacity declines as expected after adult hypoxia exposure, yet animals with prolonged postnatal hyperoxia exposure demonstrate no significant decrease in exercise capacity following adult hypoxia. Points represent individual animals, and error bars represent means ± SE. Analysis by 1-way ANOVA; **P < 0.01 compared with RA-RA; ##P < 0.01 compared with RA-H; ¤P < 0.05 for pre- and posthypoxia comparison.

Fig. 5.

Postnatal hyperoxia is associated with increased RV capillary density. RV capillary density was assessed by immunofluorescence. Wheat germ agglutinin (WGA) antibody was used to stain cell membranes (green), whereas nuclei were stained with 4′-6-diamidino-2-phenylindole (DAPI) (blue). Cardiomyocytes are identified by typical shape and myoglobin autofluorescence surrounded by WGA staining (arrows); capillaries are identified by size and autofluorescence surrounded by WGA staining (arrowheads). Cardiomyocytes and capillaries were quantified in a blinded fashion, and capillary-to-myocyte ratios were determined. Left: capillary/myocyte ratios after analysis. Right: representative images at ×40. Note increased capillarization following brief hyperoxia-hypoxia exposure. Size bars = 25 μm. Points represent individual animals, and error bars represent means ± SE. Analysis by 1-way ANOVA. *P < 0.05 compared with RA-RA. HPF, high-power field.

Fig. 6.

Postnatal hyperoxia is associated with a more robust fetal gene expression pattern. End points were determined by real-time RT-PCR of RV homogenates. Hypoxia induces a decrease in α-myosin heavy chain (α-MHC, A) and increase in β-MHC (B) gene expression consistent with a fetal gene profile. Atrial natriuretic peptide (ANP) gene expression also increases after hypoxia exposure consistent with a fetal gene profile and is potentiated in animals with a history of early hyperoxia exposure (C). Points represent individual animals, and error bars represent means ± SE. Analysis by 1-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001 compared with RA-RA.