TNIK regulation of interferon signaling and endothelial cell response to virus infection.

Background: Traf2 and Nck-interacting kinase (TNIK) is known for its regulatory role in various processes within cancer cells. However, its role within endothelial cells (ECs) has remained relatively unexplored. Methods: Leveraging RNA-seq data and Ingenuity Pathway Analysis (IPA), we probed the potential impact of TNIK depletion on ECs. Results: Examination of RNA-seq data uncovered more than 450 Differentially Expressed Genes (DEGs) in TNIK-depleted ECs, displaying a fold change exceeding 2 with a false discovery rate (FDR) below 0.05. IPA analysis unveiled that TNIK depletion leads to the inhibition of the interferon (IFN) pathway [-log (p-value) >11], downregulation of IFN-related genes, and inhibition of Hypercytokinemia/Hyperchemokinemia [-log (p-value) >8]. The validation process encompassed qRT-PCR to evaluate mRNA expression of crucial IFN-related genes, immunoblotting to gauge STAT1 and STAT2 protein levels, and ELISA for the quantification of IFN and cytokine secretion in siTNIK-depleted ECs. These assessments consistently revealed substantial reductions upon TNIK depletion. When transducing HUVECs with replication incompetent E1-E4 deleted adenovirus expressing green fluorescent protein (Ad-GFP), it was demonstrated that TNIK depletion did not affect the uptake of Ad-GFP. Nonetheless, TNIK depletion induced cytopathic effects (CPE) in ECs transduced with wild-type human adenovirus serotype 5 (Ad-WT). Summary: Our findings suggest that TNIK plays a crucial role in regulating the EC response to virus infections through modulation of the IFN pathway.

A Population of M2 Macrophages Associated With Bone Formation.

We previously identified transient brown adipocyte-like cells associated with heterotopic ossification (HO). These ancillary cells support new vessel synthesis essential to bone formation. Recent studies have shown that the M2 macrophage contributes to tissue regeneration in a similar way. To further define the phenotype of these brown adipocyte-like cells they were isolated and characterized by single-cell RNAseq (scRNAseq). Analysis of the transcriptome and the presence of surface markers specific for macrophages suggest that these cells are M2 macrophages. To validate these findings, clodronate liposomes were delivered to the tissues during HO, and the results showed both a significant reduction in these macrophages as well as bone formation. These cells were isolated and shown in culture to polarize towards either M1 or M2 similar to other macrophages. To confirm that these are M2 macrophages, mice received lipopolysacheride (LPS), which induces proinflammation and M1 macrophages. The results showed a significant decrease in this specific population and bone formation, suggesting an essential role for M2 macrophages in the production of bone. To determine if these macrophages are specific to HO, we isolated these cells using fluorescence-activated cell sorting (FACS) from a bone defect model and subjected them to scRNAseq. Surprisingly, the macrophage populations overlapped between the two groups (HO-derived versus callus) suggesting that they may be essential ancillary cells for bone formation in general and not selective to HO. Of further note, their unique metabolism and lipogenic properties suggest the potential for unique cross talk between these cells and the newly forming bone.

A replicating stem-like cell that contributes to bone morphogenetic protein 2-induced heterotopic bone formation.

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.

Characterization of neuromas in peripheral nerves and their effects on heterotopic bone formation.

The formation of neuromas involves expansion of the cellular components of peripheral nerves. The onset of these disorganized tumors involves activation of sensory nerves and neuroinflammation. Particularly problematic in neuroma is arborization of axons leading to extreme, neuropathic pain. The most common sites for neuroma are the ends of transected nerves following injury; however, this rodent model does not reliably result in neuroma formation. In this study, we established a rodent model of neuroma in which the sciatic nerve was loosely ligated with two chromic gut sutures. This model formed neuromas reliably (∼95%), presumably through activation of the neural inflammatory cascade. Resulting neuromas had a disorganized structure and a significant number of replicating cells. Quantification of changes in perineurial and Schwann cells showed a significant increase in these populations. Immunohistochemical analysis showed the presence of ß-tubulin 3 in the rapidly expanding nerve and a decrease in neurofilament heavy chain compared to the normal nerve, suggesting the axons forming a disorganized structure. Measurement of the permeability of the blood-nerve barrier shows that it opened almost immediately and remained open as long as 10 days. Studies using an antagonist of the ß3-adrenergic receptor (L-748,337) or cromolyn showed a significant reduction in tumor size and cell expansion as determined by flow cytometry, with an improvement in the animal's gait detected using a Catwalk system. Previous studies in our laboratory have shown that heterotopic ossification is also a result of the activation of neuroinflammation. Since heterotopic ossification and neuroma often occur together in amputees, they were induced in the same limbs of the study animals. More heterotopic bone was formed in animals with neuromas as compared to those without. These data collectively suggest that perturbation of early neuroinflammation with compounds such as L-748,337 and cromolyn may reduce formation of neuromas.

Cell-Based Gene Therapy System for Delivering BMPs.

The use of an adenoviral vector to transduce cells allows for certain secreted proteins or growth factors to be generated in vivo in eukaryotic cells with accurate posttranslational processing. The use of transduced cells eliminates viral toxicity, allows for targeted expression of the secreted factor at a specific site, and ensures that the therapy will be turned off when the cells are cleared by the organism. Here we describe the delivery system which utilizes cells transduced with a non-replicating adenovirus containing bone morphogenetic protein 2 (BMP-2) in the E1 region of the cassette. With this method of delivery, small amounts of the protein can incite de novo bone formation.

Trauma-Induced Heterotopic Ossification Regulates the Blood-Nerve Barrier.

De novo bone formation can occur in soft tissues as a result of traumatic injury. This process, known as heterotopic ossification (HO), has recently been linked to the peripheral nervous system. Studies suggest that HO may resemble neural crest-derived bone formation and is activated through the release of key bone matrix proteins leading to opening of the blood-nerve barrier (BNB). One of the first steps in this process is the activation of a neuro-inflammatory cascade, which results in migration of chondro-osseous progenitors, and other cells from both the endoneurial and perineurial regions of the peripheral nerves. The perineurial cells undergo brown adipogenesis, to form essential support cells, which regulate expression and activation of matrix metallopeptidase 9 (MMP9) an essential regulatory protein involved in opening the BNB. However, recent studies suggest that, in mice, a key bone matrix protein, bone morphogenetic protein 2 (BMP2) is able to immediately cross the BNB to activate signaling in specific cells within the endoneurial compartment. BMP signaling correlates with bone formation and appears critical for the induction of HO. Surprisingly, several other bone matrix proteins have also been reported to regulate the BNB, leading us to question whether these matrix proteins are important in regulating the BNB. However, this temporary regulation of the BNB does not appear to result in degeneration of the peripheral nerve, but rather may represent one of the first steps in innervation of the newly forming bone.

Is heterotopic ossification getting nervous?: The role of the peripheral nervous system in heterotopic ossification.

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".

Characterization of Brown Adipose-Like Tissue in Trauma-Induced Heterotopic Ossification in Humans.

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.

Progenitors in Peripheral Nerves Launch Heterotopic Ossification.

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.

Location-dependent heterotopic ossification in the rat model: The role of activated matrix metalloproteinase 9.

Extremity amputation or traumatic injury can often lead to the formation of heterotopic ossification (HO). Studies to induce HO in rat muscle using cell-based gene therapy show that this process appears to be location dependent. In the present study, HO was induced in mice and rats through injection of immunologically matched cells transduced with either a replication-defective adenovirus possessing bone morphogenetic protein 2 (BMP2) or an empty adenovirus vector (control). Injection in rat near the skeletal bone resulted in HO, whereas cells injected into the same muscle group but distal from the bone did not result in bone formation. When cells were injected in the same limb at both locations at the same time, HO was formed at both sites. Characterization of the bone formation in rats versus mice demonstrated that different sources of osteogenic progenitors were involved, which may account for the location dependent bone formation observed in the rat. Further experimentation has shown that a potential reason for this difference may be the inability of rat to activate matrix metalloproteinase 9 (MMP9), an essential protease in mice necessary for recruitment of progenitors. Inhibition of active MMP9 in mice led to a significant decrease in HO. The studies reported here provide insight into the mechanisms and pathways leading to bone formation in different animals and species. It appears that not all animal models are appropriate for testing HO therapies, and our studies also challenge the conventional wisdom that larger animal models are better for testing treatments affecting bone. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1894-1904, 2016.

Anaplerotic Accumulation of Tricarboxylic Acid Cycle Intermediates as Well as Changes in Other Key Metabolites During Heterotopic Ossification.

Heterotopic ossification (HO) is the de novo formation of bone that occurs in soft tissue, through recruitment, expansion, and differentiation of multiple cells types including transient brown adipocytes, osteoblasts, chondrocytes, mast cells, and platelets to name a few. Much evidence is accumulating that suggests changes in metabolism may be required to accomplish this bone formation. Recent work using a mouse model of heterotopic bone formation reliant on delivery of adenovirus-transduced cells expressing low levels of BMP2 showed the immediate expansion of a unique brown adipocyte-like cell. These cells are undergoing robust uncoupled oxidative phosphorylation to a level such that oxygen in the microenvironment is dramatically lowered creating areas of hypoxia. It is unclear how these oxygen changes ultimately affect metabolism and bone formation. To identify the processes and changes occurring over the course of bone formation, HO was established in the mice, and tissues isolated at early and late times were subjected to a global metabolomic screen. Results show that there are significant changes in both glucose levels, as well as TCA cycle intermediates. Additionally, metabolites necessary for oxidation of stored lipids were also found to be significantly elevated. The complete results of this screen are presented here, and provide a unique picture of the metabolic changes occurring during heterotopic bone formation.

Osteoblasts Have a Neural Origin in Heterotopic Ossification.

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.

Far-red fluorescence gene reporter tomography for determination of placement and viability of cell-based gene therapies.

Non-invasive injectable cellular therapeutic strategies based on sustained delivery of physiological levels of BMP-2 for spinal fusion are emerging as promising alternatives, which could provide sufficient fusion without the associated surgical risks. However, these injectable therapies are dependent on bone formation occurring only at the specific target region. In this study, we developed and deployed fluorescence gene reporter tomography (FGRT) to provide information on in vivo cell localization and viability. This information is sought to confirm the ideal placement of the materials with respect to the area where early bone reaction is required, ultimately providing three dimensional data about the future fusion. However, because almost all conventional fluorescence gene reporters require visible excitation wavelengths, current in vivo imaging of fluorescent proteins is limited by high tissue absorption and confounding autofluorescence. We previously administered fibroblasts engineered to produce BMP-2, but is difficult to determine 3-D information of placement prior to bone formation. Herein we used the far-red fluorescence gene reporter, IFP1.4 to report the position and viability of fibroblasts and developed 3-D tomography to provide placement information. A custom small animal, far-red fluorescence tomography system integrated into a commercial CT scanner was used to assess IFP1.4 fluorescence and to demark 3-D placement of encapsulated fibroblasts with respect to the vertebrae and early bone formation as assessed from CT. The results from three experiments showed that the placement of the materials within the spine could be detected. This work shows that in vivo fluorescence gene reporter tomography of cell-based gene therapy is feasible and could help guide cell-based therapies in preclinical models.

Rapid healing of femoral defects in rats with low dose sustained BMP2 expression from PEGDA hydrogel microspheres.

Current strategies for bone regeneration after traumatic injury often fail to provide adequate healing and integration. Here, we combined the poly (ethylene glycol) diacrylate (PEGDA) hydrogel with allogeneic "carrier" cells transduced with an adenovirus expressing BMP2. The system is unique in that the biomaterial encapsulates the cells, shielding them and thus suppressing destructive inflammatory processes. Using this system, complete healing of a 5 mm-long femur defect in a rat model occurs in under 3 weeks, through secretion of 100-fold lower levels of protein as compared to doses of recombinant BMP2 protein used in studies which lead to healing in 2-3 months. New bone formation was evaluated radiographically, histologically, and biomechanically at 2, 3, 6, 9, and 12 weeks after surgery. Rapid bone formation bridged the defect area and reliably integrated into the adjacent skeletal bone as early as 2 weeks. At 3 weeks, biomechanical analysis showed the new bone to possess 79% of torsional strength of the intact contralateral femur. Histological evaluation showed normal bone healing, with no infiltration of inflammatory cells with the bone being stable approximately 1 year later. We propose that these osteoinductive microspheres offer a more efficacious and safer clinical option over the use of rhBMP2.

Hyaluronan turnover and hypoxic brown adipocytic differentiation are co-localized with ossification in calcified human aortic valves.

The calcification process in aortic stenosis has garnered considerable interest but only limited investigation into selected signaling pathways. This study investigated mechanisms related to hypoxia, hyaluronan homeostasis, brown adipocytic differentiation, and ossification within calcified valves. Surgically explanted calcified aortic valves (n=14) were immunostained for markers relevant to these mechanisms and evaluated in the center (NodCtr) and edge (NodEdge) of the calcified nodule (NodCtr), tissue directly surrounding nodule (NodSurr); center and tissue surrounding small "prenodules" (PreNod, PreNodSurr); and normal fibrosa layer (CollFibr). Pearson correlations were determined between staining intensities of markers within regions. Ossification markers primarily localized to NodCtr and NodEdge, along with markers related to hyaluronan turnover and hypoxia. Markers of brown adipocytic differentiation were frequently co-localized with markers of hypoxia. In NodCtr and NodSurr, brown fat and ossification markers correlated with hyaluronidase-1, whereas these markers, as well as hypoxia, correlated with hyaluronan synthases in NodEdge. The protein product of tumor necrosis factor-α stimulated gene-6 strongly correlated with ossification markers and hyaluronidase in the regions surrounding the nodules (NodSurr, PreNodSurr). In conclusion, this study suggests roles for hyaluronan homeostasis and the promotion of hypoxia by cells demonstrating brown fat markers in calcific aortic valve disease.

Histologic identification of brown adipose and peripheral nerve involvement in human atherosclerotic vessels.

The disease mechanisms and histology of plaque development associated with atherosclerosis remain incredibly complex and not entirely understood. Recent investigations have emphasized the importance of inflammation in atherosclerosis. Several studies have also indicated heterotopic or extraskeletal bone formation in atherosclerotic vessels. The mechanisms behind heterotopic ossification appear to have similarities to those underlying atherosclerosis, with inflammation being a key inductive component to heterotopic ossification. Therefore, in the present study, we evaluated the histology associated with pathologies of atherosclerosis and heterotopic ossification in 271 coronary vessel tissue samples. We examined the prevalence and features of the inflammatory response as well as new vessel and bone formation. Inflammation and neovascularization were observed both in the adventitia and within the atherosclerotic lesions of the vessels themselves. Intriguingly, neural changes, including collections of inflammatory cells and expression of neuroinflammatory factors, were detected in the adventitial nerves of the vessels. Mature lamellar bone was found in 18 coronary vessels (7%), often with hematopoietic elements and active bone remodeling. Brown adipocytes, which pattern heterotopic bone formation, were present within the atherosclerotic lesions (28%, or 75/271). As expected, there was a strong correlation between the presence of cholesterol and plaque formation (P < .0001), but there also seemed to be a trend toward a connection between the presence of brown adipocytes and plaque. From this histologic evaluation, along with cholesterol and dystrophic calcification, we noted a novel appearance of brown adipocytes as well as neural changes, which may provide new insights to further our understanding of atherosclerosis.

Rapid Heterotrophic Ossification with Cryopreserved Poly(ethylene glycol-) Microencapsulated BMP2-Expressing MSCs.

Autologous bone grafting is the most effective treatment for long-bone nonunions, but it poses considerable risks to donors, necessitating the development of alternative therapeutics. Poly(ethylene glycol) (PEG) microencapsulation and BMP2 transgene delivery are being developed together to induce rapid bone formation. However, methods to make these treatments available for clinical applications are presently lacking. In this study we used mesenchymal stem cells (MSCs) due to their ease of harvest, replication potential, and immunomodulatory capabilities. MSCs were from sheep and pig due to their appeal as large animal models for bone nonunion. We demonstrated that cryopreservation of these microencapsulated MSCs did not affect their cell viability, adenoviral BMP2 production, or ability to initiate bone formation. Additionally, microspheres showed no appreciable damage from cryopreservation when examined with light and electron microscopy. These results validate the use of cryopreservation in preserving the viability and functionality of PEG-encapsulated BMP2-transduced MSCs.

Transient brown adipocyte-like cells derive from peripheral nerve progenitors in response to bone morphogenetic protein 2.

Perineurial-associated brown adipocyte-like cells were rapidly generated during bone morphogenetic protein 2 (BMP2)-induced sciatic nerve remodeling in the mouse. Two days after intramuscular injection of transduced mouse fibroblast cells expressing BMP2 into wild-type mice, there was replication of beta-3 adrenergic receptor(+) (ADRB3(+)) cells within the sciatic nerve perineurium. Fluorescence-activated cell sorting and analysis of cells isolated from these nerves confirmed ADRB3(+) cell expansion and their expression of the neural migration marker HNK1. Similar analysis performed 4 days after BMP2 delivery revealed a significant decrease in ADRB3(+) cells from isolated sciatic nerves, with their concurrent appearance within the adjacent soft tissue, suggesting migration away from the nerve. These soft tissue-derived cells also expressed the brown adipose marker uncoupling protein 1 (UCP1). Quantification of ADRB3-specific RNA in total hind limb tissue revealed a 3-fold increase 2 days after delivery of BMP2, followed by a 70-fold increase in UCP1-specific RNA after 3 days. Expression levels then rapidly returned to baseline by 4 days. Interestingly, these ADRB3(+) UCP1(+) cells also expressed the neural guidance factor reelin. Reelin(+) cells demonstrated distinct patterns within the injected muscle, concentrated toward the area of BMP2 release. Blocking mast cell degranulation-induced nerve remodeling resulted in the complete abrogation of UCP1-specific RNA and protein expression within the hind limbs following BMP2 injection. The data collectively suggest that local BMP2 administration initiates a cascade of events leading to the expansion, migration, and differentiation of progenitors from the peripheral nerve perineurium to brown adipose-like cells in the mouse, a necessary prerequisite for associated nerve remodeling.