Maternal Vitamin D Deficiency Does Not Cause Airway Hyperresponsiveness And Increased Airway Smooth Muscle In Young Mice

Rationale: We have recently found that vitamin D deficiency causes AHR and increased airway smooth muscle (ASM) mass, as well as reduced transforming growth factor (TGF)-β levels in the lungs of adult 8-week old female mice. We thus aimed to determine if AHR and increased ASM mass was present at 4-weeks of age and if the timing of vitamin D deficiency (i.e. in utero or postnatal deficiency) had an effect on these changes at 8-weeks of age. We also investigated if TGF-β signaling was altered due to vitamin D deficiency in 4-week old mice.

Methods: A physiologically relevant mouse model of vitamin D deficiency was developed by raising BALB/c mice on vitamin D-deficient or -replete diets. To study the effects of the timing of vitamin D deficiency, vitamin D-deficient and -replete pups were cross-fostered at birth to vitamin D-replete and -deficient mothers, respectively. Studies were carried out in 4-week old mice, and 8-week old cross-fostered mice of both sexes. AHR was assessed by measuring lung function responses to increasing doses of inhaled methacholine using the SQIREQ flexiVent system. Lungs were inflation-fixed with 4% formaldehyde and 5-micron-thick transverse sections from the left lung were stained with Masson’s Trichrome. The cross-sectional area of ASM and internal perimeter basement membrane were measured and the ASM area was then corrected for the internal perimeter. Relative gene expression of TGF-β1, TGF-β1 receptor, TGF-β2, TGF-β2 receptor, TGF-β3 and TGF-β3 was measured by real-time PCR.

Results: There were no differences in airway resistance (R ), tissue damping (G) and tissue elastance (H) at the maximal dose of aw methacholine, or ASM mass between 4-week old vitamin D-deficient and –replete mice. There were no differences in the expression of TGF-β pathway signaling molecules. Cross-fostering did not have an effect on female mice as whole-life vitamin D deficiency was required for increased R , G and H. Male mice that had postnatal vitamin D deficiency had lower R (p=0.03) and H (p=0.04) as adults compared aw with whole-life vitamin D-deficient males.

Conclusions: The increased ASM mass and AHR we previously observed in adult female mice were not apparent at 4-weeks of age. While sufficient levels of vitamin D in utero appeared to have a beneficial effect on lung function in male mice, overall the effect of vitamin D deficiency on ASM may be independent of the lung development process.