Heterotopic ossification (HO) is an aberrant regenerative process with ectopic bone induction in response to musculoskeletal trauma, in which mesenchymal stem cells (MSC) differentiate into ...osteochondrogenic cells instead of myocytes or tenocytes. Despite frequent cases of hospitalized musculoskeletal trauma, the inflammatory responses and cell population dynamics that regulate subsequent wound healing and tissue regeneration are still unclear. Here we examine, using a mouse model of trauma-induced HO, the local microenvironment of the initial post-injury inflammatory response. Single cell transcriptome analyses identify distinct monocyte/macrophage populations at the injury site, with their dynamic changes over time elucidated using trajectory analyses. Mechanistically, transforming growth factor beta-1 (TGFβ1)-producing monocytes/macrophages are associated with HO and aberrant chondrogenic progenitor cell differentiation, while CD47-activating peptides that reduce systemic macrophage TGFβ levels and help ameliorate HO. Our data thus implicate CD47 activation as a therapeutic approach for modulating monocyte/macrophage phenotypes, MSC differentiation and HO formation during wound healing.
Cells sense the extracellular environment and mechanical stimuli and translate these signals into intracellular responses through mechanotransduction, which alters cell maintenance, proliferation, ...and differentiation. Here we use a mouse model of trauma-induced heterotopic ossification (HO) to examine how cell-extrinsic forces impact mesenchymal progenitor cell (MPC) fate. After injury, single-cell (sc) RNA sequencing of the injury site reveals an early increase in MPC genes associated with pathways of cell adhesion and ECM-receptor interactions, and MPC trajectories to cartilage and bone. Immunostaining uncovers active mechanotransduction after injury with increased focal adhesion kinase signaling and nuclear translocation of transcriptional coactivator TAZ, inhibition of which mitigates HO. Similarly, joint immobilization decreases mechanotransductive signaling, and completely inhibits HO. Joint immobilization decreases collagen alignment and increases adipogenesis. Further, scRNA sequencing of the HO site after injury with or without immobilization identifies gene signatures in mobile MPCs correlating with osteogenesis, and signatures from immobile MPCs with adipogenesis. scATAC-seq in these same MPCs confirm that in mobile MPCs, chromatin regions around osteogenic genes are open, whereas in immobile MPCs, regions around adipogenic genes are open. Together these data suggest that joint immobilization after injury results in decreased ECM alignment, altered MPC mechanotransduction, and changes in genomic architecture favoring adipogenesis over osteogenesis, resulting in decreased formation of HO.
Pain is a central feature of soft tissue trauma, which under certain contexts, results in aberrant osteochondral differentiation of tissue-specific stem cells. Here, the role of sensory nerve fibers ...in this abnormal cell fate decision is investigated using a severe extremity injury model in mice. Soft tissue trauma results in NGF (Nerve growth factor) expression, particularly within perivascular cell types. Consequently, NGF-responsive axonal invasion occurs which precedes osteocartilaginous differentiation. Surgical denervation impedes axonal ingrowth, with significant delays in cartilage and bone formation. Likewise, either deletion of Ngf or two complementary methods to inhibit its receptor TrkA (Tropomyosin receptor kinase A) lead to similar delays in axonal invasion and osteochondral differentiation. Mechanistically, single-cell sequencing suggests a shift from TGFβ to FGF signaling activation among pre-chondrogenic cells after denervation. Finally, analysis of human pathologic specimens and databases confirms the relevance of NGF-TrkA signaling in human disease. In sum, NGF-mediated TrkA-expressing axonal ingrowth drives abnormal osteochondral differentiation after soft tissue trauma. NGF-TrkA signaling inhibition may have dual therapeutic use in soft tissue trauma, both as an analgesic and negative regulator of aberrant stem cell differentiation.
Myeloid cells are critical to the development of fibrosis following muscle injury; however, the mechanism of their role in fibrosis formation remains unclear. In this study, we demonstrate that ...myeloid cell-derived TGF-β1 signaling is increased in a profibrotic ischemia reperfusion and cardiotoxin muscle injury model. We found that myeloid-specific deletion of
abrogates the fibrotic response in this injury model and reduces fibro/adipogenic progenitor cell proliferation while simultaneously enhancing muscle regeneration, which is abrogated by adaptive transfer of normal macrophages. Similarly, a murine TGFBRII-Fc ligand trap administered after injury significantly reduced muscle fibrosis and improved muscle regeneration. This study ultimately demonstrates that infiltrating myeloid cell TGF-β1 is responsible for the development of traumatic muscle fibrosis, and its blockade offers a promising therapeutic target for preventing muscle fibrosis after ischemic injury.
Our objective was to identify macrophage subpopulations and gene signatures associated with regenerative or fibrotic healing across different musculoskeletal injury types.
Subpopulations of ...macrophages are hypothesized to fine tune the immune response after damage, promoting either normal regenerative, or aberrant fibrotic healing.
Mouse single-cell RNA sequencing data before and after injury were assembled from models of musculoskeletal injury, including regenerative and fibrotic mouse volumetric muscle loss (VML), regenerative digit tip amputation, and fibrotic heterotopic ossification. R packages Harmony , MacSpectrum , and Seurat were used for data integration, analysis, and visualizations.
There was a substantial overlap between macrophages from the regenerative VML (2 mm injury) and regenerative bone models, as well as a separate overlap between the fibrotic VML (3 mm injury) and fibrotic bone (heterotopic ossification) models. We identified 2 fibrotic-like (FL 1 and FL 2) along with 3 regenerative-like (RL 1, RL 2, and RL 3) subpopulations of macrophages, each of which was transcriptionally distinct. We found that regenerative and fibrotic conditions had similar compositions of proinflammatory and anti-inflammatory macrophages, suggesting that macrophage polarization state did not correlate with healing outcomes. Receptor/ligand analysis of macrophage-to-mesenchymal progenitor cell crosstalk showed enhanced transforming growth factor β in fibrotic conditions and enhanced platelet-derived growth factor signaling in regenerative conditions.
Characterization of macrophage subtypes could be used to predict fibrotic responses following injury and provide a therapeutic target to tune the healing microenvironment towards more regenerative conditions.
Although midlobular hepatocytes in zone 2 are a recently identified cellular source for liver homeostasis and regeneration, these cells have not been exclusively fate mapped. We generated an ...Igfbp2-CreER knockin strain that specifically labels midlobular hepatocytes. During homeostasis over 1 year, zone 2 hepatocytes increased in abundance from occupying 21%–41% of the lobular area. After either pericentral injury with carbon tetrachloride or periportal injury with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), IGFBP2+ cells replenished lost hepatocytes in zones 3 and 1, respectively. IGFBP2+ cells also preferentially contributed to regeneration after 70% partial hepatectomy, as well as liver growth during pregnancy. Because IGFBP2 labeling increased substantially with fasting, we used single nuclear transcriptomics to explore zonation as a function of nutrition, revealing that the zonal division of labor shifts dramatically with fasting. These studies demonstrate the contribution of IGFBP2-labeled zone 2 hepatocytes to liver homeostasis and regeneration.
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•Igfbp2-CreER mice allow for genetic manipulation in midlobular zone 2 hepatocytes•Lineage tracing showed that midlobular hepatocytes proliferate during homeostasis•Zone 2 cells were protected from common liver injuries and replaced lost hepatocytes•Fasting induced significant changes in liver zonation
The liver consists of different zones with spatial heterogeneity in their metabolic functions. Here, a new Igfbp2-CreER line, which enabled direct tracing of midlobular hepatocytes, shows that IGFBP2+ cells serve as a source of new hepatocytes during normal homeostasis and regeneration.
Heterotopic ossification (HO), the formation of ectopic bone in soft tissues, has been extensively studied in its two primary forms: post-traumatic HO (tHO) typically found in patients who have ...experienced musculoskeletal or neurogenic injury and in fibrodysplasia ossificans progressiva (FOP), where it is genetically driven. Given that in both diseases HO arises via endochondral ossification, the molecular mechanisms behind both diseases have been postulated to be manifestations of similar pathways including those activated by BMP/TGFβ superfamily ligands. A significant step towards understanding the molecular mechanism by which HO arises in FOP was the discovery that FOP causing ACVR1 variants trigger HO in response to activin A, a ligand that does not activate signaling from wild type ACVR1, and that is not inherently osteogenic in wild type settings. The physiological significance of this finding was demonstrated by showing that activin A neutralizing antibodies stop HO in two different genetically accurate mouse models of FOP. In order to explore the role of activin A in tHO, we performed single cell RNA sequencing and compared the expression of activin A as well as other BMP pathway genes in tHO and FOP HO. We show that activin A is expressed in response to injury in both settings, but by different types of cells. Given that wild type ACVR1 does not transduce signal when engaged by activin A, we hypothesized that inhibition of activin A will not block tHO. Nonetheless, as activin A was expressed in tHO lesions, we tested its inhibition and compared it with inhibition of BMPs. We show here that anti-activin A does not block tHO, whereas agents such as antibodies that neutralize ACVR1 or ALK3-Fc (which blocks osteogenic BMPs) are beneficial, though not completely curative. These results demonstrate that inhibition of activin A should not be considered as a therapeutic strategy for ameliorating tHO.
Heterotopic ossification (HO) is a challenging condition that occurs after musculoskeletal injury and is characterized by the formation of bone in non-skeletal tissues. While the effect of HO on ...blood vessels is well established, little is known about its impact on lymphatic vessels. Here, we use a mouse model of traumatic HO to investigate the relationship between HO and lymphatic vessels. We show that injury triggers lymphangiogenesis at the injury site, which is associated with elevated vascular endothelial growth factor C (VEGF-C) levels. Through single-cell transcriptomic analyses, we identify mesenchymal progenitor cells and tenocytes as sources of Vegfc. We demonstrate by lineage tracing that Vegfc-expressing cells undergo osteochondral differentiation and contribute to the formation of HO. Last, we show that Vegfc haploinsufficiency results in a nearly 50% reduction in lymphangiogenesis and HO formation. These findings shed light on the complex mechanisms underlying HO formation and its impact on lymphatic vessels.
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•HO formation induces lymphangiogenesis within the injured tissue•MPCs and tenocytes express the lymphangiogenic factor Vegfc.•Vegfc lineage cells expand after injury and contribute to the formation of cartilage and HO
Vishlaghi et al. investigate lymphangiogenesis during heterotopic ossification (HO). Their study reveals that injury triggers lymphangiogenesis within the affected tissue. Furthermore, they demonstrate that Vegfc-lineage progenitor cells undergo altered cell fate, ultimately contributing to HO. These findings offer crucial insights into the involvement of lymphatic vessels in HO formation.
The liver is remarkably regenerative and can completely recover even when 80% of its mass is surgically removed. Identification of secreted factors that regulate liver growth would help us understand ...how organ size and regeneration are controlled but also provide candidate targets to promote regeneration or impair cancer growth.
To enrich for secreted factors that regulate growth control, we induced massive liver overgrowth with either YAP or MYC . Differentially expressed secreted factors were identified in these livers using transcriptomic analysis. To rank candidates by functionality, we performed in vivo CRISPR screening using the Fah knockout model of tyrosinemia. We identified secreted phosphoprotein-2 (SPP2) as a secreted factor that negatively regulates regeneration. Spp2 -deficient mice showed increased survival after acetaminophen poisoning and reduced fibrosis after repeated carbon tetrachloride injections. We examined the impact of SPP2 on bone morphogenetic protein signaling in liver cells and found that SPP2 antagonized bone morphogenetic protein signaling in vitro and in vivo. We also identified cell-surface receptors that interact with SPP2 using a proximity biotinylation assay coupled with mass spectrometry. We showed that SPP2's interactions with integrin family members are in part responsible for some of the regeneration phenotypes.
Using an in vivo CRISPR screening system, we identified SPP2 as a secreted factor that negatively regulates liver regeneration. This study provides ways to identify, validate, and characterize secreted factors in vivo.
Monitoring wound progression over time is a critical aspect for studies focused on in-depth molecular analysis or on evaluating the efficacy of potential novel therapies. Histopathological analysis ...of wound biopsies can provide significant insight into healing dynamics, yet there is no standardized and reproducible scoring system currently available. The purpose of this study was to develop and statistically validate a scoring system based on parameters in each phase of healing that can be easily and accurately assessed using either Hematoxylin & Eosin (H&E) or Masson's Trichrome (MT) staining. These parameters included re-epithelization, epithelial thickness index, keratinization, granulation tissue thickness, remodeling, and the scar elevation index. The initial phase of the study was to (1) optimize and clarify healing parameters to limit investigator bias and variability; (2) compare the consistency of parameters assessed using H&E versus MT staining. During the validation phase of this study, the accuracy and reproducibility of this scoring system was independently iterated upon and validated in four different types of murine skin wound models (Excisional; punch biopsy; pressure ulcers; burn wounds). A total of
= 54 histology sections were randomized, blinded, and assigned to two groups of independent investigators (
= 5 per group) for analysis. The sensitivity of each parameter (ranging between 80% and 95%) is reported with illustrations on the appropriate assessment method using ImageJ software. In the validated scoring system, the lowest score (score:0) is associated with an open/unhealed wound as is evident immediately and within the first day postinjury, whereas the highest score (score:12) is associated with a completely closed and healed wound without excessive scarring. This study defines and describes the minimum recommended criteria for assessing wound healing dynamics using the SPOT skin wound score. The acronym SPOT refers to the academic and scientific institutions that were involved in the development of the scoring system, namely, Stellenbosch University, Polish Academy of Sciences, Obatala Sciences, and the University of Texas Southwestern.
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