Elodie Baptista (1), Lina Tabcheh (1), Juliana Marulanda Montoya (2, Chaohua Deng (1), Joseph Deering (3), Jean-Yves Jouzeau (1), Arnaud Bianchi (1), Marc D. McKee (3), Monzur Murshed (2), Hervé Kempf (1)
Affiliation(s):
1. UMR 7365 CNRS-Université de Lorraine, Ingénierie Moléculaire, cellulaire et Physiopathologie, Vandœuvre-lès-Nancy, France
2. Department of Medicine, and Faculty of Dental Medicine and Oral Health Sciences, McGill University, Montreal, QC, Canada
3. Faculty of Dental Medicine and Oral Health Sciences, and Department of Anatomy and Cell Biology, McGill University, Montreal, QC, Canada
Introduction: Tracheal calcification is a rare condition mostly found in the elderly where tracheal elasticity is compromised, leading eventually to dyspnea. Tracheal calcification is also found in younger individuals suffering from pathologic conditions such as Keutel Syndrome (KS), a rare genetic disease caused by loss-of-function mutations in the Matrix Gla Protein (MGP) gene. MGP is a calcification inhibitor, and KS patients show abnormal apatitic mineral deposition in most of the cartilaginous tissues, which is accompanied by midface hypoplasia, brachytelephalangism, peripheral pulmonary stenosis and respiratory complications including asthma, dyspnea and respiratory tract infection. In this context, we sought to understand the mechanisms lying at the origin of tracheal mineralization that so far have been unexplored, and more specifically to investigate the role of MGP in this process.
Material & Methods: Alcian blue / Alizarin red and Von Kossa / Alcian blue double stainings were performed respectively on whole trachea and on 5-µm thick sections of trachea samples at different postnatal ages (P0 to P60) in wild-type (WT, Mgp+/+) and in MGP-deficient (Mgp-/-) mice. Collagen X expression was detected by in situ hybridization in cartilage rings and also assessed by qPCR. Sub-micron micro-computed X-ray tomography (µCT), also known as X-ray Microscopy (Zeiss Xradia Versa) from trachea of WT and knockout mice was also performed and analyzed using Dragonfly software.
Results: Our morphological and histological investigations revealed an unexpected phenomenon in WT mice, wherein tracheal calcification occurred as early as 30 days after birth. With aging, this mineralization extended in a rostro-caudal pattern along the respiratory tract, through a process involving terminal differentiation of tracheal chondrocytes overexpressing the hypertrophic marker collagen X. In MGP-deficient mice, the overall mineralization of the respiratory tract was accelerated, with calcification of tracheal cartilage rings starting at 14 days after birth. Moreover, Mgp-/- mice also exhibited an unexpected calcification in the tracheal mucosa, more precisely in the lamina propria, associated with a structural modification of the overlying epithelium. This calcification was restricted to the trachea and was not found in other epithelia in these knockout mice. No similar lamina propria calcification was observed in WT mice, even in very old mice (up to 24 months) when all tracheal cartilage rings are calcified.
Conclusion: The present study is the first to comprehensively describe mouse tracheal mineralization and provide evidence that calcification of the cartilage rings appears to be a sudden and early physiologic event in mice. It also demonstrates that MGP is a key factor regulating mineralization of the tracheobronchial tree. Indeed, MGP deficiency accelerates tracheal cartilage mineralization and appears responsible for an additional calcification of the trachea in the mucosal layer, which leads to changes that could explain the respiratory problems found in patients with KS who suffer from dyspnea, cough, stridor and infections (previously attributed to tracheal ring calcification).
Keywords
Trachea ; Calcification ; Matrix Gla Protein