Chronic hypercapnia alters lung matrix composition in mouse pups

Abstract

Rationale: permissive hypercapnia, a stretch-limiting ventilation strategy, often results in high Pa(CO(2)). This strategy is associated with reduced morbidity and mortality in premature infants and its benefits have been attributed to diminished barotrauma. However, little is known about the independent effect of high CO(2) levels during the lung development.

Methods: mice were exposed to 8% CO(2) or room air for 2 wk either from postnatal day 2 through 17 or as adults (approximately 2 mo of age). Lungs were excised and processed for protein, RNA, histology, and total lung volumes.

Results: histologic analysis demonstrated that alveolar walls of CO(2)-exposed mouse pups were thinner than those of controls and had twice the total lung volume. Molecular analysis revealed that several matrix proteins in the lung were downregulated in mouse pups exposed to hypercapnia. Interstitial collagen type I alpha1, type III alpha1, elastin and fibronectin protein, and mRNA levels were less than half of controls while collagen IV alpha 5 was unaffected. This decrease in interstitial collagen could thus account for the thinning of the interstitial matrix and the altered lung biomechanics. Matrix metalloproteinase (MMP)-8, a collagenase that has specificity for collagen types I and III, increased in hypercapnic mouse pups, suggesting increased collagen degradation. Moreover, tissue inhibitor of MMP (TIMP)-1, a potent inhibitor of MMP-8, was significantly decreased. However, unlike pups, adult mice exposed to hypercapnia demonstrated only a mild increase in total lung volumes and did not exhibit similar molecular or histologic changes.

Conclusions: although permissive hypercapnia may prevent lung injury from barotrauma, our study revealed that exposure to hypercapnia may be an important factor in lung remodeling and function, especially in early life.

Fig. 1.

A: 2-wk exposure to 8% CO₂ results in decreased alveolar interstitial thickness without changing mean linear intercept (×20 magnification). B: ×40 magnification of 1-μm-thick sections demonstrate that interstitial spaces between alveoli are thinner in the CO₂ group.

Fig. 2.

Static lung volumes at 30 cmH₂0 pressure (representing total lung volume) demonstrate an increase in lung volume in mice exposed to chronic hypercapnia, n ≥ 8 for each group.

Fig. 3.

Collagen was significantly reduced in the CO₂ group compared controls using a Sircol Collagen Assay. Collagen was reduced by 14% in the experimental group (1,311 vs. 1,125 μg collagen/g lung, P = 0.03, n = 8 each group).

Fig. 4.

A: representative Western blots collagen types I α1, III α1, IV α5, and VI α1, fibronectin, elastin, α smooth muscle actin, and Hsc-70. B: densitometry of Western blots demonstrates a reduction of collagen types I α1, III α1, and VI α1, whereas collagen type IV α5 was unaltered. Fibronectin and elastin levels were also significantly reduced but CO₂ did not affect alpha smooth muscle actin protein levels, n ≥ 6 each group.

Fig. 5.

A: representative Western blots illustrate that active MMP-8 levels increase with hypercapnia, whereas TIMP-1 protein levels are significantly decreased. Densitometry of Western blots demonstrate significance at P = 0.01 for both proteins, n ≥ 6 for each group. B: TIMP-1 ELISA assay also confirms the reduction of function in the CO₂ group, P = 0.001, n = 5.

Fig. 6.

A: adult mice exposed to a 2-week exposure to 8% CO₂ did not demonstrate a significant change in lung morphology (×20 magnification with a ×40 insert). B: densitometry of Western blots did not demonstrate a significant difference between adult control and experimental groups for collagen types I α1. III α1, or MMP-8 protein levels, n ≥ 5 for each group, P > 0.05. C: static lung volumes at 30 cmH₂O pressure (representing total lung volume) demonstrate that adult mice exposed to hypercapnia only demonstrated a 15% increase in lung volume (P = 0.05), whereas pups exposed to the same conditions increased 40% (P < 0.002) from age matched controls, n ≥ 8 for pup groups and n = 4 for adult groups.