Studies presented here, using a murine model of bone morphogenetic protein type 2 (BMP2)‐induced heterotopic ossification (HO) show that the protein initiates HO by signaling through progenitors in ...the endoneurium of peripheral nerves. In the mouse, these cells were identified in the endoneurium one day after BMP2 induction using antibody against phosphoSMAD (PS) 1, 5, and 8. Studies conducted in a tracking mouse that contains a tamoxifen‐regulated Wnt1‐Cre recombinase crossed with a td Tomato red (TR) reporter (Wnt1CreErt:Ai9Tm) confirmed their neural origin. In this model both BMP2 induction and tamoxifen are absolutely required to induce TR. SP7+(osterix+)TR+ cells were found in the endoneurium on day 1 and associated with bone on day 7. Quantification of TR+ and TR− cells isolated by fluorescence‐activated cell sorting showed that all SP7+ cells were found in the TR+ population, whereas only about 80% of the TR+ cells expressed SP7. Pre‐chondrocytes (Sox 9+) and transient brown fat (tBAT, UCP1+) also coexpressed TR, suggesting that the progenitor in nerves is multi‐potential. The endoneurium of human nerves near the site of HO contained many PS+ cells, and SP7+ cells were found in nerves and on bone in tissue from patients with HO. Control tissues and nerves did not contain these PS+ and SP7+ cells. Some osteoblasts on bone from patients with HO were positive for PS, suggesting the continued presence of BMP during bone formation. The data suggests that the progenitors for HO are derived from the endoneurium in both the mouse model of HO and in humans with HO. Stem Cells Translational Medicine 2017;6:1109–1119
Bone morphogenetic protein 2 (BMP2)‐induced heterotopic bone formation (HBF) starts synchronously from zero upon BMP2 induction, which is advantageous for lineage tracking. The studies reported here ...in GLAST‐CreErt2:tdTomato red (TR)floxSTOPflox mice during BMP2‐induced HBF show 78.8 ± 11.6% of chondrocytes and 86.5 ± 1.9% of osteoblasts are TR+ after approximately 1 week. Clustering after single‐cell RNAseq resulted in nine cell types, and analysis revealed one as a highly replicating stem‐like cell (RSC). Pseudotiming suggested that the RSC transitions to a mesenchymal stem‐like cell that simultaneously expresses multiple osteoblast and chondrocyte transcripts (chondro‐osseous progenitor COP). RSCs and COPs were isolated using flow cytometry for unique surface markers. Isolated RSCs (GLAST‐TR+ Hmmr+ Cd200−) and COPs (GLAST‐TR+ Cd200+ Hmmr−) were injected into the muscle of mice undergoing HBF. Approximately 9% of the cells in heterotopic bone (HB) in mice receiving RSCs were GLAST‐TR+, compared with less than 0.5% of the cells in mice receiving COPs, suggesting that RSCs are many times more potent than COPs. Analysis of donor‐derived TR+ RSCs isolated from the engrafted HB showed approximately 50% were COPs and 45% were other cells, presumably mature bone cells, confirming the early nature of the RSCs. We next isolated RSCs from these mice (approximately 300) and injected them into a second animal, with similar findings upon analysis of HBF. Unlike other methodology, single cell RNAseq has the ability to detect rare cell populations such as RSCs. The fact that RSCs can be injected into mice and differentiate suggests their potential utility for tissue regeneration.
Blue arrows show the trajectory of heterotopic bone formation after bone morphogenetic protein 2 induction. The replicating stem‐like cell (RSC) is an epithelial cell that undergoes an epithelial‐mesenchymal transition in forming the chondro‐osseous progenitor (COP) that expresses both osteoblast and chondrocyte transcripts. Red arrows show the fluorescence‐activated cell sorting isolation of either TR+ RSCs or TR+ COPs and the injection of 3000 of each TR+ cell into wild‐type mice undergoing heterotopic ossification.
Osteoprotegerin (OPG), also known as osteoclastogenesis inhibitory factor or tumor necrosis factor receptor superfamily member 11B, is well known as a modulator of bone remodeling. The contribution ...of OPG to cardiovascular disease (CVD) has been suggested, but its molecular mechanism is complex and remains unclear. In the present study, Alves-Lopes et al. (Clin. Sci. (Lond.) (2021) 135(20): https://doi.org/10.1042/CS20210643) reported the critical role of syndecan-1 (SDC-1, also known as CD138), a surface protein part of the endothelial glycocalyx, in OPG-induced vascular dysfunction. The authors found that in endothelial cells (ECs), through SDC-1, OPG increased eNOS Thr495 phosphorylation, thereby inhibiting eNOS activity. Furthermore, the OPG-SDC-1 interaction increased reactive oxygen species (ROS) production through NOX1/4 activation. Both the reduced eNOS activity and induced ROS production inhibited NO production and impaired EC function. In vascular smooth muscle cells (VSMCs), the OPG-SDC-1 interaction increased ROS production through NOX1/4 activation, subsequently increased MLC phosphorylation-mediated Rho kinase-MYPT1 regulation, leading to increased vascular contraction. Ultilizing wire myography and mechanistic studies, the authors nicely provide the evidence that SDC-1 plays a crucial role in OPG-induced vascular dysfunction. As we mentioned above, the molecular mechanism and roles of OPG in cardiovascular system are complex and somewhat confusing. In this commentary, we briefly summarize the OPG-mediated signaling pathways in cardiovascular system.
Heterotopic ossification (HO), or de novo bone formation in soft tissue, is often observed following traumatic injury. Recent studies suggest that peripheral nerves may play a key functional role in ...this process. The results supporting a neurological basis for HO are examined in this article. Evidence supports the fact that BMPs released from bone matrix possess the capacity to induce HO. However, the process cannot be recapitulated using recombinant proteins without extremely high doses suggesting other components are required for this process. Study of injuries that increase risk for HO, i.e. amputation, hip replacement, elbow fracture, burn, and CNS injury suggests that a likely candidate is traumatic injury of adjacent peripheral nerves. Recent studies suggest neuroinflammation may play a key functional role, by its ability to open the blood-nerve barrier (BNB). Barrier opening is characterized by a change in permeability and is experimentally assessed by the ability of Evans blue dye to enter the endoneurium of peripheral nerves. A combination of BMP and barrier opening is required to activate bone progenitors in the endoneurial compartment. This process is referred to as “neurogenic HO”.
Overview of neurogenic heterotopic ossification (HO). Induction of HO is thought to occur from the release of BMPs in bone matrix that enter through the blood nerve barrier, presumably due to selective permeability associated with specific injuries. Once inside, BMP2 induces neuroinflammation through activation of the TRVP1 receptor leading to the release of substance P and CGRP. This leads to the recruitment and degranulation of mast cells outside the barrier that can eventually activate MMP9 and other proteins involved in matrix remodeling, which open the barrier and allow cells to exit through the circulation. Parallel to this process, cells within the endoneurium undergo early osteogenic differentiation and express factors necessary for extravasation to the site of HO. As they exit the barrier to be deposited, cells within the perineurium have been activated through the release of noradrenaline and subsequent binding to the β3-adrenergic receptor. These cells have an immediate expansion of their mitochondria and undergo robust uncoupled aerobic respiration similar to brown adipose. These cells start to migrate towards the site of HO, where their elevated oxygen consumption leads to both localized tissue hypoxia potentially necessary to support not only chondrogenesis, but also activation of HIF1α pathways that support new vessel formation. These cells express VEGFA, necessary for patterning the location of new vessels. The uncoupled respiration also appears to be able to coordinately regulate sensory nerves to maintain this process. However, when the inductive components, such as BMPs are gone, the process terminates, suggesting that they are transient in nature. Display omitted
•Damage to the bone from traumatic injury may release small amounts of BMPs that change the kinetics, leading to HO.•Damage to nerves disrupts the blood- nerve barrier by causing it to open in regions near to the injury site.•Neural progenitor cells, essential for HO, exit the nerve and enter the circulation before going to the site of HO.•Perineural brown-adipocyte-like cells migrate to the site of HO and function to form vessels and pattern bone.
Background
Heterotopic ossification (HO) is the process of bone formation at a nonskeletal site. Recently, we showed that the earliest steps occur in sensory nerves. We now extend these studies by ...identifying unique osteogenic progenitors within the endoneurial compartment of sensory nerves.
Questions/purposes
We asked: (1) What is the nature of the osteoprogenitor in the endoneurium of peripheral nerves? (2) How do osteoprogenitors travel from the nerve to the site of new bone formation?
Methods
HO was induced by intramuscular injection of Ad5BMP-2-transduced cells in mice. Osteoprogenitors were identified through immunohistochemistry and then quantified and further characterized by fluorescence-activated cell sorting and immunocytochemistry. The kinetics of the appearance of markers of extravasation was determined by quantitative reverse transcription-polymerase chain reaction. In each experiment mice were injected with bone morphogenetic protein-2 (BMP-2)-producing cells (experimental) or with cells transduced with empty vector or, in some cases, a group receiving no injection (control).
Results
Induction of HO leads to the expression, within 24 hours, of osteoblast-specific transcription factors in cells in the endoneurium followed by their coordinate disappearance from the nerve at 48 hours. They reappear in blood also at 48 hours after induction. During vessel entrance they begin to express the tight junction molecule, claudin 5. The cells expressing both the osteoblast-specific transcription factor, osterix, as well as claudin 5, then disappear from circulation at approximately 3 to 4 days by extravasation into the site of new bone formation. These endoneurial osteoprogenitors express neural markers PDGFRα, musashi-1, and the low-affinity nerve growth factor receptor p75(NTR) as well as the endothelial marker Tie-2. In a key experiment, cells that were obtained from mice that were injected with cells transduced with an empty vector, at 2 days after injection, contained 0.83% (SD, 0.07; 95% confidence interval CI, 0.59–1.05) cells expressing claudin 5. However, cells that were obtained from mice 2 days after injection of BMP-2-producing cells contained 4.5% cells expressing claudin 5 (SD, 0.72%; 95% CI, 2.01–6.94; p < 0.0015). Further analysis revealed that all of the cells expressing claudin 5 were found to be positive for osteoblast-specific markers, whereas cells not expressing claudin 5 were negative for these same markers.
Conclusions
The findings suggest that the endoneurial progenitors are the major osteogenic precursors that are used for HO. They exit the nerve through the endoneurial vessels, flow through vessels to the site of new bone formation, and then extravasate out of the vessels into this site.
Clinical Relevance
The biogenesis of osteoblasts in HO is very different than expected and shows that HO is, at least in part, a neurological disorder. This could result in a major shift in orthopaedic methodologies to prevent or treat this disease. The fact that nerves are intimately involved in the process may also provide clues that will lead to an explanation of the clinical fact that HO often occurs as a result of traumatic brain injury.
ERK5 (extracellular signal-regulated kinase 5) is a dual kinase transcription factor containing an N-terminal kinase domain and a C-terminal transcriptional activation domain. Many ERK5 kinase ...inhibitors have been developed and tested to treat cancer and inflammatory diseases. However, recent data have raised questions about the role of the catalytic activity of ERK5 in proliferation and inflammation. We aimed to investigate how ERK5 reprograms myeloid cells to the proinflammatory senescent phenotype, subsequently leading to atherosclerosis.
A ERK5 S496A (dephosphorylation mimic) knock in (KI) mouse model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9), and atherosclerosis was characterized by hypercholesterolemia induction. The plaque phenotyping in homozygous ERK5 S496A KI and wild type (WT) mice was studied using imaging mass cytometry. Bone marrow-derived macrophages were isolated from hypercholesterolemic mice and characterized using RNA sequencing and functional in vitro approaches, including senescence, mitochondria reactive oxygen species, and inflammation assays, as well as by metabolic extracellular flux analysis.
We show that atherosclerosis was inhibited in ERK5 S496A KI mice. Furthermore, ERK5 S496 phosphorylation mediates both senescence-associated secretory phenotype and senescence-associated stemness by upregulating AHR (aryl hydrocarbon receptor) in plaque and bone marrow-derived macrophages isolated from hypercholesterolemic mice. We also discovered that ERK5 S496 phosphorylation could induce NRF2 (NFE2-related factor 2) SUMOylation at a novel K518 site to inhibit NRF2 transcriptional activity without altering ERK5 catalytic activity and mediates oxidized LDL (low-density lipoprotein)-induced senescence-associated secretory phenotype. Specific ERK5 kinase inhibitors (AX15836 and XMD8-92) also inhibited ERK5 S496 phosphorylation, suggesting the involvement of ERK5 S496 phosphorylation in the anti-inflammatory effects of these ERK5 kinase inhibitors.
We discovered a novel mechanism by which the macrophage ERK5-NRF2 axis develops a unique senescence-associated secretory phenotype/stemness phenotype by upregulating AHR to engender atherogenesis. The finding of senescence-associated stemness phenotype provides a molecular explanation to resolve the paradox of senescence in proliferative plaque by permitting myeloid cells to escape the senescence-induced cell cycle arrest during atherosclerosis formation.
Heterotopic ossification (HO), the abnormal formation of bone within soft tissues, is a major complication after severe trauma or amputation. Transient brown adipocytes have been shown to be a ...critical regulator of this process in a mouse model of HO. In this study, we evaluated the presence of brown fat within human HO lesions. Most of the excised tissue samples displayed histological characteristics of bone, fibroproliferative cells, blood vessels, and adipose tissue. Immunohistochemical analysis revealed extensive expression of uncoupling protein 1 (UCP1), a definitive marker of brown adipocytes, within HO-containing tissues but not normal tissues. As seen in the brown adipocytes observed during HO in the mouse, these UCP1+ cells also expressed the peroxisome proliferator-activated receptor γ coactivator 1α. However, further characterization showed these cells, like their mouse counterparts, did not express PR domain containing protein 16, a key factor present in brown adipocytes found in depots. Nor did they express factors present in beige adipocytes. These results identify a population of UCP1+ cells within human tissue undergoing HO that do not entirely resemble either classic brown or beige adipocytes, but rather a specialized form of brown adipocyte-like cells, which have a unique function. These cells may offer a new target to prevent this unwanted bone.
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