Elucidating the Role of Type I Collagen Mutations on Respiratory Function in Osteogenesis Imperfecta

Roy Morello, PhD

I am a musculoskeletal researcher with an active interest in rare diseases, especially those caused by collagen alterations. Our disease of choice for the last 20 years has been osteogenesis imperfecta (brittle bone disease) that is most commonly caused by alterations in type I collagen. Its severity ranges from mild to lethal and it dramatically affects the skeleton by causing low bone mass and recurrent fractures after minimal trauma.

In addition to studying the impact of this disease on the skeleton, about 7 years ago—in collaboration with John L. Carroll, MD, a pulmonologist at Arkansas Children’s Hospital—we began looking into its impact on the lung and respiration. In fact, OI can cause severe respiratory distress at birth, and adult patients with OI often suffer from decreasing pulmonary function and are more vulnerable to diseases such as pneumonia.

Premise

The role of congenital collagen alterations during lung development and their effect on lung structure and function are not well understood. To study these important aspects, we elected to use a rare disease such as OI as the paradigm of a connective tissue disease with a primary alteration in type I collagen and as an entry point into a poorly addressed but medically relevant issue.

OI, caused by either dominant mutations in COL1A1 and COL1A2 genes or by loss of function of genes involved in type I collagen processing (e.g. CRTAP) is the most common bone fragility disorder;^1,2 however, respiratory distress at birth and a decline in respiratory function are leading causes of mortality in patients with OI.³ This is because type I collagen is expressed in most tissues and the disease is systemic, with skeletal and extra-skeletal manifestations that affect many organs, including the lung.

Therefore, there is a critical need to understand how type I collagen alterations negatively impact lung development and function with the goal to identify new therapeutic targets to improve the health of patients with OI.

Earlier findings

We recently demonstrated that, similar to what happens in osteoblasts and skin fibroblasts, the production of type I collagen is also dysregulated in lung fibroblasts in the CrtapKO mouse model of OI.⁴ We have now analyzed 3 distinct mouse models of OI and identified intrinsic lung defects consistent with early onset impairment of alveolar formation that led to significant alterations in respiratory function by 3 months of age.⁵ This contradicts the current model that predicts that the skeletal features of the OI disease, such as abnormalities of the chest wall and kyphoscoliosis are the only causes that prevent proper lung inflation leading to restrictive disease. Interestingly, mouse models mimicking a more severe type of OI showed more severe changes in their respiratory parameters.⁵

Objective and hypothesis

The overall objective of this proposal is to gain adequate mechanistic understanding to dissect and separate the contribution of intrinsic developmental lung changes from those of extrinsic skeletal changes causing chest wall hypoplasia or defects. Our central hypothesis is that intrinsic lung changes contribute significantly to respiratory impairment in OI and result in cellular loss during the terminal morphogenetic process of alveolar formation.

Aims

We proposed to determine the consequences of type I collagen mutations in the lung independent from their effects on the skeleton, using a newly developed conditional knock-in mouse model for type I collagen (aim 1); we also proposed to identify cellular and molecular changes in the lung of CrtapKO compared to WT mice using single-cell RNA sequencing (aim 2).

Results

The lack of a mouse model to study the effects of OI type I collagen mutations in a tissue-specific manner has prevented us to understand the primary impact of these mutations on the respiratory system because the skeletal changes caused by OI (in the thoracic cage, vertebral column, ribs, and/or diaphragm) can significantly affect lung function. To overcome this long-standing limitation, we generated a new mouse model where a classic glycine substitution (p.G1146R) in the triple helical region of type I collagen can be expressed in specific cells/tissue using a mouse Cre-recombinase driver strain. We crossed this new mouse model with a transgenic mouse expressing Cre in all tissues and confirmed that the offspring carrying both the new allele (Col1a1G1146RFloxed/+) and the Cre-recombinase reproduced all features of a severe form of OI, including low bone mass and fractures but also severe alveolar simplification in the distal lung parenchyma together with alterations in several respiratory mechanic parameters.

When we expressed the Col1a1 glycine substitution just in the lung (using a Tbx4-Cre mouse strain), a maneuver that leaves the skeleton intact, our data indicated that the parenchyma morphology of the lung was mostly intact and several of the respiratory mechanics parameters were normal and undistinguishable from the control mice, unlike what we observed when expressing the mutation globally. However, both the K parameter (measuring the concavity of the descending limb of the PV curve) and V10_TLC (the lung volume at 10 cmH20 expressed as a percentage of the total lung capacity) were still significantly reduced in Col1a1G1146RFloxed/+;Tbx4-Cre+ mice compared to controls.

These data indicate that the expression of the G1146R mutation in the lung only causes a milder phenotype. However, the significantly reduced K and V10_TLC parameters indicate intrinsic lung changes consistent perhaps with potential fibrosis and/or surfactant alterations.

Collectively, our data suggest that skeletal defects in OI play a major impact on respiratory function of OI patients. However, it is important to remember that type I collagen is also expressed in the diaphragm and the pleural membranes and its alterations in these structures may also significantly contribute to altered lung development/function in OI.

We are also pursuing studies to determine cellular and gene expression changes in a recessive mouse model of OI (Crtap-/- mice). We have currently performed 2 independent experiments using single-cell RNA sequencing on WT and Crtap-/- 5-week-old mice, and plan to perform a third one soon. We are observing changes in fibroblast cell populations in the lung and the third experiment will be useful to determine whether such changes also impact alveolar epithelial cells.

The analysis of these data is still ongoing, and we will also hopefully delineate transcriptional changes affecting the critical process of alveologenesis. Thanks to the American Lung Association support, we have now secured additional funding from the National Institutes of Health (NIH) to pursue and further expand these studies using spatial transcriptomics.

Future

Newborns diagnosed with a skeletal dysplasia, including OI, frequently suffer from respiratory problems that result in prolonged and expensive hospital stays in pediatric intensive care units with limited therapeutic options. These patients offer unique challenges for the physician due to the complex care they require. It is our hope that our studies will lead to a better understanding of the role of type I collagen in alveologenesis and provide new therapeutic targets for improved lung disease management in patients with OI.

References

1. ^{Marini, J.C., Forlino, A., Bachinger, H.P., Bishop, N.J., Byers, P.H., Paepe, A., Fassier, F., Fratzl-Zelman, N., Kozloff, K.M., Krakow, D., et al. 2017. Osteogenesis imperfecta. Nat Rev Dis Primers 3:17052.}
2. ^{Jovanovic, M., and Marini, J.C. 2024. Update on the Genetics of Osteogenesis Imperfecta. Calcif Tissue Int.}
3. ^{Folkestad, L., Hald, J.D., Canudas-Romo, V., Gram, J., Hermann, A.P., Langdahl, B., Abrahamsen, B., and Brixen, K. 2016. Mortality and Causes of Death in Patients With Osteogenesis Imperfecta: A Register-Based Nationwide Cohort Study. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.}
4. ^{Dimori, M., Heard-Lipsmeyer, M.E., Byrum, S.D., Mackintosh, S.G., Kurten, R.C., Carroll, J.L., and Morello, R. 2020. Respiratory defects in the CrtapKO mouse model of osteogenesis imperfecta. American journal of physiology. Lung cellular and molecular physiology 318:L592-L605.}
5. ^{Dimori, M., Fett, J., Leikin, S., Otsuru, S., Thostenson, J.D., Carroll, J.L., and Morello, R. 2022. Distinct type I collagen alterations cause intrinsic lung and respiratory defects of variable severity in mouse models of osteogenesis imperfecta. The Journal of physiology.}