Circulating TGF-β Pathway in Osteogenesis Imperfecta Pediatric Patients Subjected to MSCs-Based Cell Therapy

Arantza Infante, Leire Cabodevilla, Blanca Gener, Clara I Rodríguez, Arantza Infante, Leire Cabodevilla, Blanca Gener, Clara I Rodríguez

Abstract

Osteogenesis Imperfecta (OI) is a rare genetic disease characterized by bone fragility, with a wide range in the severity of clinical manifestations. The majority of cases are due to mutations in COL1A1 or COL1A2, which encode type I collagen. There is no cure for OI, and real concerns exist for current therapeutic approaches, mainly antiresorptive drugs, regarding their effectiveness and security. Safer and effective therapeutic approaches are demanded. Cell therapy with mesenchymal stem cells (MSCs), osteoprogenitors capable of secreting type I collagen, has been tested to treat pediatric OI with encouraging outcomes. Another therapeutic approach currently under clinical development focuses on the inhibition of TGF-β pathway, based on the excessive TGF-β signaling found in the skeleton of severe OI mice models, and the fact that TGF-β neutralizing antibody treatment rescued bone phenotypes in those OI murine models. An increased serum expression of TGF-β superfamily members has been described for a number of bone pathologies, but still it has not been addressed in OI patients. To delve into this unexplored question, in the present study we investigated serum TGF-β signalling pathway in two OI pediatric patients who participated in TERCELOI, a phase I clinical trial based on reiterative infusions of MSCs. We examined not only the expression and bioactivity of circulating TGF-β pathway in TERCELOI patients, but also the effects that MSCs therapy could elicit. Strikingly, basal serum from the most severe patient showed an enhanced expression of several TGF-β superfamily members and increased TGF-β bioactivity, which were modulated after MSCs therapy.

Keywords: TGF-β; cell therapy; mesenchymal stem cells; osteogenesis imperfecta; stem cells.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2022 Infante, Cabodevilla, Gener and Rodríguez.

Figures

FIGURE 1
FIGURE 1
Schematic diagram illustrating TERCELOI clinical trial cellular infusions and the current study of circulating TGF-β expression and bioactivity in patients’ sera collected before (basal serum) and after the five consecutive MSCs infusions. The post-infusions sera included those collected 1 week (1w), 1 month (1 m) and 4 months (4 m) after each cell infusion, as well as the follow-up sera, collected 1 year and 2 years after the last cell infusion.
FIGURE 2
FIGURE 2
Basal circulating TGF-β superfamily members’ expression and bioactivity in OI patients. (A) Left heatmap shows the fluorescent signal intensities for target proteins expressed in P01 and P02 basal sera samples. Only the spots with a fluorescent intensity cutoff ≥300 above the background were considered. Right heatmap magnified shows the fluorescent intensities of the TGF-β superfamily members present in basal P01 and P02 sera are shown (right heatmap). (B) Graph showing the total number of expressed proteins in P01 and P02 basal sera, grouped according to their fluorescent signal intensity: low (300-2,000), medium (2,000-10,000) and high (>10,000). (C) Left, schematic representation of the HEK-Blue-TGF-βTM reporter cell line assay. Right, Scatter plot with bars showing TGF-β-induced SEAP in HEK-Blue-TGF-βTM cells. The TGF-β induced SEAP from patients’ sera samples is shown normalized versus that shown by the negative control (C-; w/o TGF-β). C+ stands for the positive control, cells stimulated with TGF-β (10 ng/ml). The experiments were performed in octuplicates for each condition and repeated two independent times. Data are mean ± standard deviation obtained from the two independent experiments. Each dot represents an independent experiment.
FIGURE 3
FIGURE 3
Circulating TGF-β superfamily members expression and bioactivity after MSCs therapy in OI patients. (A) Heatmap showing the expression ratio of TGF-β superfamily members after the 1st MSCs infusion in P01 and P02, calculated as the fluorescent signal intensity of target proteins after the MSCs therapy, 1 week (1w), 1 month (1 m) or 4 months (4 m) versus the fluorescent intensity of target proteins from each patient’s basal serum. To be considered, a cutoff ratio of ±1.5 was established. (B) Scatter plot showing TGF-β induced SEAP of HEK-Blue-TGF-βTM cells exposed to P01 and P02 sera collected after the first MSCs infusion and compared to the results obtained with basal serum for each patient. The TGF-β induced SEAP is shown normalized versus the negative control. (C) Scatter plot showing TGF-β induced SEAP of HEK-Blue-TGF-βTM cells exposed to the sera from P01 obtained during the 2nd, 3rd, 4th, 5th infusions and follow-up visits, and compared to the results obtained with basal serum. Two independent experiments were performed with P01 and P02 sera, each of one with their respective positive (c+) and negative controls (c-). Within each independent experiment, each condition was performed in octuplicates. Data are mean ± standard deviation obtained from the two independent experiments. Each dot represents an independent experiment.Dashed horizontal lines illustrate the TGF-β bioactivity level of basal P01 and P02 sera.

References

    1. Faraji A., Abtahi S., Ghaderi A., Samsami Dehaghani A. (2016). Transforming Growth Factor β1 (TGF-Β1) in the Sera of Postmenopausal Osteoporotic Females. Int. J. Endocrinol. Metab. 14 (4), e36511. 10.5812/ijem.36511
    1. Franken R., Den Hartog A. W., De Waard V., Engele L., Radonic T., Lutter R., et al. (2013). Circulating Transforming Growth Factor-β as a Prognostic Biomarker in Marfan Syndrome. Int. J. Cardiol. 168 (3), 2441–2446. 10.1016/j.ijcard.2013.03.033
    1. Gebken J., Brenner R., Feydt A., Notbohm H., Brinckmann J., Müller P. K., et al. (2000). Increased Cell Surface Expression of Receptors for Transforming Growth Factor-β on Osteoblasts from Patients with Osteogenesis Imperfecta. Pathobiology 68 (3), 106–112. 10.1159/000055910
    1. Götherström C., Westgren M., Shaw S. W., Aström E., Biswas A., Byers P. H., et al. (2014). Pre- and Postnatal Transplantation of Fetal Mesenchymal Stem Cells in Osteogenesis Imperfecta: a Two-center Experience. Stem Cell Transl Med 3 (2), 255–264. 10.5966/sctm.2013-0090
    1. Grafe I., Yang T., Alexander S., Homan E. P., Lietman C., Jiang M. M., et al. (2014). Excessive Transforming Growth Factor-β Signaling Is a Common Mechanism in Osteogenesis Imperfecta. Nat. Med. 20 (6), 670–675. 10.1038/nm.3544
    1. Grainger D. J., Percival J., Chiano M., Spector T. D. (1999). The Role of Serum TGF-β Isoforms as Potential Markers of Osteoporosis. Osteoporos. Int. 9 (5), 398–404. 10.1007/s001980050163
    1. Greene B., Russo R. J., Dwyer S., Malley K., Roberts E., Serrielo J., et al. (2021). Inhibition of TGF-β Increases Bone Volume and Strength in a Mouse Model of Osteogenesis Imperfecta. JBMR Plus 5 (9), e10530. 10.1002/jbm4.10530
    1. Hering S., Isken E., Knabbe C., Janott J., Jost C., Pommer A., et al. (2001). TGFbeta1 and TGFbeta2 mRNA and Protein Expression in Human Bone Samples. Exp. Clin. Endocrinol. Diabetes 109 (4), 217–226. 10.1055/s-2001-15109
    1. Hering S., Jost C., Schulz H., Hellmich B., Schatz H., Pfeiffer H. (2002). Circulating Transforming Growth Factor Beta1 (TGFbeta1) Is Elevated by Extensive Exercise. Eur. J. Appl. Physiol. 86 (5), 406–410. 10.1007/s00421-001-0537-5
    1. Horwitz E. M., Gordon P. L., Koo W. K. K., Marx J. C., Neel M. D., Mcnall R. Y., et al. (2002). Isolated Allogeneic Bone Marrow-Derived Mesenchymal Cells Engraft and Stimulate Growth in Children with Osteogenesis Imperfecta: Implications for Cell Therapy of Bone. Proc. Natl. Acad. Sci. 99 (13), 8932–8937. 10.1073/pnas.132252399
    1. Horwitz E. M., Prockop D. J., Fitzpatrick L. A., Koo W. W. K., Gordon P. L., Neel M., et al. (1999). Transplantability and Therapeutic Effects of Bone Marrow-Derived Mesenchymal Cells in Children with Osteogenesis Imperfecta. Nat. Med. 5 (3), 309–313. 10.1038/6529
    1. Infante A., Gener B., Vázquez M., Olivares N., Arrieta A., Grau G., et al. (2021). Reiterative Infusions of MSCs Improve Pediatric Osteogenesis Imperfecta Eliciting a Pro-osteogenic Paracrine Response: TERCELOI Clinical Trial. Clin. Transl Med. 11 (1), e265. 10.1002/ctm2.265
    1. Jovanovic M., Guterman-Ram G., Marini J. C. (2021). Osteogenesis Imperfecta: Mechanisms and Signaling Pathways Connecting Classical and Rare OI Types. Endocr. Rev. 43 (1), 61–90. 10.1210/endrev/bnab017
    1. MacFarlane E. G., Haupt J., Dietz H. C., Shore E. M. (2017). TGF-β Family Signaling in Connective Tissue and Skeletal Diseases. Cold Spring Harb Perspect. Biol. 9 (11), a022269. 10.1210/endrev/bnab017
    1. Maioli M., Gnoli M., Boarini M., Tremosini M., Zambrano A., Pedrini E., et al. (2019). Genotype-phenotype Correlation Study in 364 Osteogenesis Imperfecta Italian Patients. Eur. J. Hum. Genet. 27 (7), 1090–1100. 10.1038/s41431-019-0373-x
    1. Marini J. C., Forlino A., Bächinger H. P., Bishop N. J., Byers P. H., Paepe A. D., et al. (2017). Osteogenesis Imperfecta. Nat. Rev. Dis. Primers 3, 17052. 10.1038/nrdp.2017.52
    1. Marini J. C., Forlino A., Cabral W. A., Barnes A. M., San Antonio J. D., Milgrom S., et al. (2007). Consortium for Osteogenesis Imperfecta Mutations in the Helical Domain of Type I Collagen: Regions Rich in Lethal Mutations Align with Collagen Binding Sites for Integrins and Proteoglycans. Hum. Mutat. 28 (3), 209–221. 10.1002/humu.20429
    1. Martinez-Hackert E., Sundan A., Holien T. (2021). Receptor Binding Competition: A Paradigm for Regulating TGF-β Family Action. Cytokine Growth Factor. Rev. 57, 39–54. 10.1016/j.cytogfr.2020.09.003
    1. Matt P., Schoenhoff F., Habashi J., Holm T., Van Erp C., Loch D., et al. (2009). Circulating Transforming Growth Factor-β in Marfan Syndrome. Circulation 120 (6), 526–532. 10.1161/circulationaha.108.841981
    1. Nickel J., Ten Dijke P., Mueller T. D. (2018). TGF-β Family Co-receptor Function and Signaling. Acta Biochim. Biophys. Sin (Shanghai) 50 (1), 12–36. 10.1093/abbs/gmx126
    1. Otsuru S., Desbourdes L., Guess A. J., Hofmann T. J., Relation T., Kaito T., et al. (2018). Extracellular Vesicles Released from Mesenchymal Stromal Cells Stimulate Bone Growth in Osteogenesis Imperfecta. Cytotherapy 20 (1), 62–73. 10.1016/j.jcyt.2017.09.012
    1. Pereira R. F., Halford K. W., O'hara M. D., Leeper D. B., Sokolov B. P., Pollard M. D., et al. (1995). Cultured Adherent Cells from Marrow Can Serve as Long-Lasting Precursor Cells for Bone, Cartilage, and Lung in Irradiated Mice. Proc. Natl. Acad. Sci. 92 (11), 4857–4861. 10.1073/pnas.92.11.4857
    1. Sarahrudi K., Thomas A., Mousavi M., Kaiser G., Köttstorfer J., Kecht M., et al. (2011). Elevated Transforming Growth Factor-Beta 1 (TGF-Β1) Levels in Human Fracture Healing. Injury 42 (8), 833–837. 10.1016/j.injury.2011.03.055
    1. Takahashi K., Akatsu Y., Podyma-Inoue K. A., Matsumoto T., Takahashi H., Yoshimatsu Y., et al. (2020). Targeting All Transforming Growth Factor-β Isoforms with an Fc Chimeric Receptor Impairs Tumor Growth and Angiogenesis of Oral Squamous Cell Cancer. J. Biol. Chem. 295 (36), 12559–12572. 10.1074/jbc.ra120.012492
    1. Tauer J. T., Abdullah S., Rauch F. (2018). Effect of Anti‐TGF‐β Treatment in a Mouse Model of Severe Osteogenesis Imperfecta. J. Bone Miner Res. 34 (2), 207–214. 10.1002/jbmr.3617
    1. Tauer J. T., Robinson M. E., Rauch F. (2019). Osteogenesis Imperfecta: New Perspectives from Clinical and Translational Research. JBMR Plus 3 (8), e10174. 10.1002/jbm4.10174
    1. Wang X., Li F., Xie L., Crane J., Zhen G., Mishina Y., et al. (2018). Inhibition of Overactive TGF-β Attenuates Progression of Heterotopic Ossification in Mice. Nat. Commun. 9 (1), 551. 10.1038/s41467-018-02988-5
    1. Wang Y., Chen Q., Zhao M., Walton K., Harrison C., Nie G. (2017). Multiple Soluble TGF-β Receptors in Addition to Soluble Endoglin Are Elevated in Preeclamptic Serum and They Synergistically Inhibit TGF-β Signaling. J. Clin. Endocrinol. Metab. 102 (8), 3065–3074. 10.1210/jc.2017-01150
    1. Wang Z., Li Y., Hou B., Pronobis M. I., Wang M., Wang Y., et al. (2020). An Array of 60,000 Antibodies for Proteome-Scale Antibody Generation and Target Discovery. Sci. Adv. 6 (11), eaax2271. 10.1126/sciadv.aax2271
    1. Wu X. Y., Peng Y. Q., Zhang H., Xie H., Sheng Z. F., Luo X. H., et al. (2013). Relationship between Serum Levels of OPG and TGF- β with Decreasing Rate of BMD in Native Chinese Women. Int. J. Endocrinol. 2013, 727164. 10.1155/2013/727164
    1. Zhen G., Wen C., Jia X., Li Y., Crane J. L., Mears S. C., et al. (2013). Inhibition of TGF-β Signaling in Mesenchymal Stem Cells of Subchondral Bone Attenuates Osteoarthritis. Nat. Med. 19 (6), 704–712. 10.1038/nm.3143
    1. Zimmermann G. G., Kusswetter M., Küsswetter A., Moghaddam S., Wentzensen A., Richter W., et al. (2005). TGF-β1 as a Marker of Delayed Fracture Healingg. Bone 36 (5), 779–785. 10.1016/j.bone.2005.02.011

Source: PubMed

Sponsors and Collaborators

Medical Conditions

Drug Interventions

3
Subscribe