SP03. Polyamine Metabolism In Mesenchymal Progenitor Cells Modulates Osteogenic Differentiation
Year: 2024
Type: article
Abstract: Purpose: Traumatic heterotopic ossification (HO) is the formation of ectopic bone in the soft tissues of the musculoskeletal system following burn and blast wounds. It is well known that tissue-resident mesenchymal progenitor cells (MPCs) aberrantly differentiate into osteogenic and chondrogenic lineages following injury. However, little is known about how cellular metabolism is altered in these pathologic progenitor cells in response to injury. Methods: Wild-type C57B6 mice received a 30% total body surface area burn injury to the dorsum and a concurrent Achilles tenotomy or no injury (n=5/group). Achilles injury site tissue or uninjured Achilles tendon was isolated seven days post-injury and digested for liquid chromatography-mass spectrometry (LC-MS) screening. Differential metabolite concentration analyses were performed on the 299 metabolites identified in tissues between injured and uninjured conditions. Previously published single-cell RNA sequencing (scRNA-seq) analyses and single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) of uninjured tendon and day 7 and 42 post-burn/tenotomy injury were also analyzed for genes in the polyamine metabolism pathway (GEO: GSE150995). Cells were harvested 7 days following tissue injury, expanded into 10 cm culture plates, and seeded into 24-well plates. Plates were treated with either culture media (DMEM+10% fetal bovine serum+1% penicillin/streptomycin, osteogenic differentiation media (ODM, DMEM+10mM beta-glycerophosphate+300uM ascorbic acid-2-phosphate), and 1% dimethyl sulfoxide solvent (DMSO), or ODM with either 25, 100, or 250 ug/ml trans-4-methylcyclohexylamine (4MCHA, inhibitor of spermidine synthase) diluted in DMSO. RNA was isolated from wells for quantitative polymerase chain reaction (qPCR) analysis of osteogenic differentiation genes or alkaline phosphatase staining for calcium deposition. Results: Metabolomics screening showed significantly higher concentrations of polyamine pathway metabolites putrescine and spermidine (ranked 1st and 3rd, respectively) in injured tissue (BT) compared to uninjured control (Un.) (A-B). When comparing all metabolites within the pathway, ornithine, putrescine, and spermidine are significantly upregulated after burn/tenotomy (C). Next, analysis of our scRNA-seq dataset showed that genes of polyamine pathway enzymes ornithine decarboxylase (Odc1), spermidine synthase (Srm), and spermine (Sms) were upregulated in mesenchymal progenitors following injury (D), suggesting that flux through this pathway is increased. Additionally, snATAC-seq showed open chromatin regions of these genes at all three time points, further supporting active transcription of these genes (not shown). To study how polyamines affect osteogenic differentiation, mesenchymal progenitor cells isolated from the tendon injury site were treated with a spermidine synthase inhibitor (4MCHA). Cells treated with 4MCHA exhibited a significant reduction of osteogenic differentiation measured by the relative expression of key osteogenic transcription factors Runx2 and Osx compared to vehicle control (E). Treated cells showed significantly reduced osteogenic differentiation by alkaline phosphatase staining one week following the osteogenic differentiation induction (F). These findings suggest that inhibiting polyamine metabolism inhibits osteogenic differentiation of HO-forming MPCs in vitro. Conclusion: These findings suggest that MPC polyamine metabolism may be necessary for post-traumatic heterotopic ossification and MPC osteogenic differentiation. 4MCHA represents a potential future metabolic-based therapy to mitigate HO.
Institution: The University of Texas Southwestern Medical Center
Language: English
Subfield: Rheumatology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $2,195
OpenAlex ID: W4394964965