Endothelin-1 mediates hypoxia-induced increases in vascular collagen in the newborn mouse lung

Abstract

Endothelin-1 (ET-1) mediates hypoxia-mediated pulmonary vascular remodeling (HPVR), and endothelin-A receptor (ET-AR) blockade prevents HPVR in newborn mice. Our objective was to determine postnatal effects of chronic hypoxia and/or ET-AR blockade on lung ET-1, ET-AR, ET-BR, and vascular collagen and elastin. Newborn C57BL/6 mice (n = 6-8/gp) given either BQ610 (ET-AR blocker) or vehicle were exposed to air or hypoxia (12% O2) from birth for 1, 3, or 14 d. Lung ET-1 was assessed by ELISA, and ET-AR and ET-BR by immunohistochemistry. Vascular collagen and elastin were assessed by quantitative image analysis. ET-1, ET-AR, ET-BR, collagen I and III, and tropoelastin mRNA levels were assessed by real-time quantitative RT-PCR. We observed that: 1) hypoxia attenuated the normal postnatal decrease in ET-1 and collagen content; 2) ET-AR blockade reduced collagen independent of O2; 3) hypoxia increased elastin mRNA expression and attenuated the normal postnatal decrease in elastin content; and 4) BQ610 reduced elastin mRNA but not elastin content. We conclude that, in neonatal mice, hypoxia attenuates normal postnatal decreases in ET-1, vascular collagen, and elastin. ET-AR blockade reduces collagen fiber area but not mRNA, and does not decrease elastin despite reducing its expression.

Figure 1

Effects of 1 and 14 d hypoxic exposure (12% O₂, 1 atm) on (A) ET-1 protein and steady-state mRNA expression, (B) ET-AR protein and steady-state mRNA expression, and (C) ET-BR protein and steady-state mRNA expression in neonatal mouse lung. Animals exposed to air or hypoxia for 14 d were administered either vehicle or BQ-610 (ET-AR antagonist) daily from birth. Results are means ± SE, n = 6–8 mice/group. *p < 0.05 vs respective air control groups; †p < 0.05 vs respective 14-d groups; ‡p < 0.05 vs respective vehicle controls.

Figure 2

Effects of hypoxia and/or BQ610 on immunohistochemical staining for ET-AR in neonatal mouse lungs after 1 and 14 d of hypoxic exposure. (400×; calibration bar = 50 μm.)

Figure 3

Effects of hypoxia and/or BQ610 on immunohistochemical staining for ET-BR in neonatal mouse lungs after 1 and 14 d of hypoxic exposure. (400×; calibration bar = 50 μm.)

Figure 4

Effects of hypoxia and/or BQ610 on perivascular collagen staining in neonatal mouse lung after 1 and 14 d of hypoxic exposure. Lung sections were stained with picrosirius red (PASR) for collagen. (A) Representative photomicrographs of pulmonary arteries (400×; calibration bar = 50 μm). (B) Total lung collagen by Sircol method, and vascular collagen area by quantitative image analysis of PASR stained sections. Results are means ± SE, n = 120–160 pulmonary arteries from 6 to 8 mice/group. *p < 0.05 vs respective air control groups, †p < 0.05 vs respective 14-d groups, ‡p < 0.05 vs respective vehicle controls.

Figure 5

Effects of hypoxia and/or BQ610 on vascular elastin staining and elastin mRNA expression on pulmonary arteries in neonatal mouse lung after 1 and 14 d of hypoxic exposure. Lung sections were stained with Verhoeff’s elastic stain for elastin. (A) Representative photomicrographs of pulmonary arteries (400×; calibration bar = 50 μm). (B) Vascular elastin area in pulmonary arteries was measured by quantitative image analysis in elastin-stained lung sections. Results are means ± SE, n = 120–160 pulmonary arteries from 6 to 8 mice/group. *p < 0.05 vs respective air control groups, †p < 0.05 vs respective 14-d groups, ‡p < 0.05 vs respective vehicle controls.