Joshua Peterson

Inhibition of Hif1α prevents both trauma-induced and genetic heterotopic ossification

Significance Heterotopic ossification (HO) is a debilitating condition in which bone forms inappropriately within soft tissues. Two vastly different patient populations are at risk for developing HO: those with musculoskeletal trauma or severe burns and those with a genetic mutation in the bone morphogenetic protein receptor ACVR1 (Activin type 1 receptor). In this study, we demonstrate that both forms of HO share a common signaling pathway through hypoxia inducible factor-1α, and that pharmacologic inhibition or genetic knockout of this signaling pathway can mitigate and even abolish HO formation. These findings pave the way for pharmacologic inhibitors of hypoxia inducible factor-1α as therapeutic options for heterotopic ossification. Pathologic extraskeletal bone formation, or heterotopic ossification (HO), occurs following mechanical trauma, burns, orthopedic operations, and in patients with hyperactivating mutations of the type I bone morphogenetic protein receptor ACVR1 (Activin type 1 receptor). Extraskeletal bone forms through an endochondral process with a cartilage intermediary prompting the hypothesis that hypoxic signaling present during cartilage formation drives HO development and that HO precursor cells derive from a mesenchymal lineage as defined by Paired related homeobox 1 (Prx). Here we demonstrate that Hypoxia inducible factor-1α (Hif1α), a key mediator of cellular adaptation to hypoxia, is highly expressed and active in three separate mouse models: trauma-induced, genetic, and a hybrid model of genetic and trauma-induced HO. In each of these models, Hif1α expression coincides with the expression of master transcription factor of cartilage, Sox9 [(sex determining region Y)-box 9]. Pharmacologic inhibition of Hif1α using PX-478 or rapamycin significantly decreased or inhibited extraskeletal bone formation. Importantly, de novo soft-tissue HO was eliminated or significantly diminished in treated mice. Lineage-tracing mice demonstrate that cells forming HO belong to the Prx lineage. Burn/tenotomy performed in lineage-specific Hif1α knockout mice (Prx-Cre/Hif1αfl:fl) resulted in substantially decreased HO, and again lack of de novo soft-tissue HO. Genetic loss of Hif1α in mesenchymal cells marked by Prx-cre prevents the formation of the mesenchymal condensations as shown by routine histology and immunostaining for Sox9 and PDGFRα. Pharmacologic inhibition of Hif1α had a similar effect on mesenchymal condensation development. Our findings indicate that Hif1α represents a promising target to prevent and treat pathologic extraskeletal bone.

Scleraxis‐Lineage Cells Contribute to Ectopic Bone Formation in Muscle and Tendon

The pathologic development of heterotopic ossification (HO) is well described in patients with extensive trauma or with hyperactivating mutations of the bone morphogenetic protein (BMP) receptor ACVR1. However, identification of progenitor cells contributing to this process remains elusive. Here we show that connective tissue cells contribute to a substantial amount of HO anlagen caused by trauma using postnatal, tamoxifen‐inducible, scleraxis‐lineage restricted reporter mice (Scx‐creERT2/tdTomatofl/fl). When the scleraxis‐lineage is restricted specifically to adults prior to injury marked cells contribute to each stage of the developing HO anlagen and coexpress markers of endochondral ossification (Osterix, SOX9). Furthermore, these adult preinjury restricted cells coexpressed mesenchymal stem cell markers including PDGFRα, Sca1, and S100A4 in HO. When constitutively active ACVR1 (caACVR1) was expressed in scx‐cre cells in the absence of injury (Scx‐cre/caACVR1fl/fl), tendons and joints formed HO. Postnatal lineage‐restricted, tamoxifen‐inducible caACVR1 expression (Scx‐creERT2/caACVR1fl/fl) was sufficient to form HO after directed cardiotoxin‐induced muscle injury. These findings suggest that cells expressing scleraxis within muscle or tendon contribute to HO in the setting of both trauma or hyperactive BMP receptor (e.g., caACVR1) activity. Stem Cells 2017;35:705–710

Local and Circulating Endothelial Cells Undergo Endothelial to Mesenchymal Transition (EndMT) in Response to Musculoskeletal Injury

Endothelial-to-mesenchymal transition (EndMT) has been implicated in a variety of aberrant wound healing conditions. However, unambiguous evidence of EndMT has been elusive due to limitations of in vitro experimental designs and animal models. In vitro experiments cannot account for the myriad ligands and cells which regulate differentiation, and in vivo tissue injury models may induce lineage-independent endothelial marker expression in mesenchymal cells. By using an inducible Cre model to mark mesenchymal cells (Scx-creERT/tdTomato + ) prior to injury, we demonstrate that musculoskeletal injury induces expression of CD31, VeCadherin, or Tie2 in mesenchymal cells. VeCadherin and Tie2 were expressed in non-endothelial cells (CD31−) present in marrow from uninjured adult mice, thereby limiting the specificity of these markers in inducible models (e.g. VeCadherin- or Tie2-creERT). However, cell transplantation assays confirmed that endothelial cells (ΔVeCadherin/CD31+/CD45−) isolated from uninjured hindlimb muscle tissue undergo in vivo EndMT when transplanted directly into the wound without intervening cell culture using PDGFRα, Osterix (OSX), SOX9, and Aggrecan (ACAN) as mesenchymal markers. These in vivo findings support EndMT in the presence of myriad ligands and cell types, using cell transplantation assays which can be applied for other pathologies implicated in EndMT including tissue fibrosis and atherosclerosis. Additionally, endothelial cell recruitment and trafficking are potential therapeutic targets to prevent EndMT.

Characterization of Heterotopic Ossification Using Radiographic Imaging: Evidence for a Paradigm Shift.

Heterotopic ossification (HO) is the growth of extra-skeletal bone which occurs following trauma, burns, and in patients with genetic bone morphogenetic protein (BMP) receptor mutations. The clinical and laboratory evaluation of HO is dependent on radiographic imaging to identify and characterize these lesions. Here we show that despite its inadequacies, plain film radiography and single modality microCT continue to serve as a primary method of HO imaging in nearly 30% of published in vivo literature. Furthermore, we demonstrate that detailed microCT analysis is superior to plain film and single modality microCT radiography specifically in the evaluation of HO formed through three representative models due to its ability to 1) define structural relationships between growing extra-skeletal bone and normal, anatomic bone, 2) provide accurate quantification and growth rate based on volume of the space-occupying lesion, thereby facilitating assessments of therapeutic intervention, 3) identify HO at earlier times allowing for evaluation of early intervention, and 4) characterization of metrics of bone physiology including porosity, tissue mineral density, and cortical and trabecular volume. Examination of our trauma model using microCT demonstrated two separate areas of HO based on anatomic location and relationship with surrounding, normal bone structures. Additionally, microCT allows HO growth rate to be evaluated to characterize HO progression. Taken together, these data demonstrate the need for a paradigm shift in the evaluation of HO towards microCT as a standard tool for imaging.

Surgical Excision of Heterotopic Ossification Leads to Re‐Emergence of Mesenchymal Stem Cell Populations Responsible for Recurrence

Trauma‐induced heterotopic ossification (HO) occurs after severe musculoskeletal injuries and burns, and presents a significant barrier to patient rehabilitation. Interestingly, the incidence of HO significantly increases with repeated operations and after resection of previous HO. Treatment of established heterotopic ossification is challenging because surgical excision is often incomplete, with evidence of persistent heterotopic bone. As a result, patients may continue to report the signs or symptoms of HO, including chronic pain, nonhealing wounds, and joint restriction. In this study, we designed a model of recurrent HO that occurs after surgical excision of mature HO in a mouse model of hind‐limb Achilles’ tendon transection with dorsal burn injury. We first demonstrated that key signaling mediators of HO, including bone morphogenetic protein signaling, are diminished in mature bone. However, upon surgical excision, we have noted upregulation of downstream mediators of osteogenic differentiation, including pSMAD 1/5. Additionally, surgical excision resulted in re‐emergence of a mesenchymal cell population marked by expression of platelet‐derived growth factor receptor‐α (PDGFRα) and present in the initial developing HO lesion but absent in mature HO. In the recurrent lesion, these PDGFRα+ mesenchymal cells are also highly proliferative, similar to the initial developing HO lesion. These findings indicate that surgical excision of HO results in recurrence through similar mesenchymal cell populations and signaling mechanisms that are present in the initial developing HO lesion. These results are consistent with findings in patients that new foci of ectopic bone can develop in excision sites and are likely related to de novo formation rather than extension of unresected bone. Stem Cells Translational Medicine 2017;6:799–806

mTOR inhibition and BMP signaling act synergistically to reduce muscle fibrosis and improve myofiber regeneration

Muscle trauma is highly morbid due to intramuscular scarring, or fibrosis, and muscle atrophy. Studies have shown that bone morphogenetic proteins (BMPs) reduce muscle atrophy. However, increased BMP signaling at muscle injury sites causes heterotopic ossification, as seen in patients with fibrodysplasia ossificans progressiva (FOP), or patients with surgically placed BMP implants for bone healing. We use a genetic mouse model of hyperactive BMP signaling to show the development of intramuscular fibrosis surrounding areas of ectopic bone following muscle injury. Rapamycin, which we have previously shown to eliminate ectopic ossification in this model, also eliminates fibrosis without reducing osteogenic differentiation, suggesting clinical value for patients with FOP and with BMP implants. Finally, we use reporter mice to show that BMP signaling is positively associated with myofiber cross-sectional area. These findings underscore an approach in which 2 therapeutics (rapamycin and BMP ligand) can offset each other, leading to an improved outcome.

Lymphatic Contribution to the Cellular Niche in Heterotopic Ossification.

Objective: The objective of this study was to determine the contribution of lymphatic tissue to heterotopic ossification (HO). Background: HO is the pathologic development of ectopic bone within soft tissues often following severe trauma. Characterization of the tissue niche supporting HO is critical to identifying therapies directed against this condition. Lymphangiogenesis is upregulated during incidents of trauma, thereby coincident with the niche supportive of HO. We hypothesized that lymphatic tissues play a critical role in HO formation. Methods: Mice underwent hindlimb Achilles’ tendon transection and dorsal burn injury (burn/tenotomy) to induce HO. The popliteal and inguinal lymph nodes were excised ipsilateral to the tenotomy site. Flow cytometry and immunostaining were used to quantify and localize lymphoendothelium. MicroCT was used to quantify HO. Results: Enrichment of mature lymphatic tissues was noted 2 weeks after injury at the tendon transection sites when compared with the contralateral, intact tendon based on LYVE1+ tubules (10.9% vs 0.8%, P < 0.05). Excision of the inguinal and popliteal nodes with draining popliteal lymphatic vessel significantly decreased the presence of mature lymphoendothelium 2 weeks after injury (10.9% vs 3.3%, P < 0.05). Bone-cartilage-stromal progenitor cells (CD105+/AlphaV+/Tie2-/CD45-/CD90-/BP1-) were also significantly decreased after lymph node excision (10.2% vs 0.5%, P < 0.05). A significant decrease was noted in the volume of de novo HO present within the soft tissues (0.12 mm3 vs 0.02 mm3). Conclusion: These findings suggest that lymphatic vessels are intimately linked with the de novo formation bone within soft tissues following trauma, and their presence may facilitate bone formation.

Risk of peritoneal dialysis catheter-associated peritonitis following kidney transplant

Peritoneal dialysis (PD) patients have equivalent or slightly better kidney transplant outcomes when compared to hemodialysis (HD) patients. However, given the risk for postoperative infection, we sought to determine the risk factors for PD catheter‐associated infections for patients who do not have the PD catheter removed at the time of engraftment.

Incidental anatomic finding of celiacomesenteric trunk associated with ‘nutcracker phenomenon,’ or compression of the left renal vein

Patient: Female, 91 Final Diagnosis: Nutcracker syndrome • celiacomesenteric trunk Symptoms: Dyspepsia • dysphagia Medication: — Clinical Procedure: — Specialty: Surgery Objective: Congenital defects/diseases Background: Celiacomesenteric trunk (CMT) is a very rare anatomic finding in which the celiac artery and the superior mesenteric artery (SMA) originate from the abdominal aorta through a common trunk. Clinical associations with CMT include arterial aneurysm, thrombosis, and celiac artery compression. However, an association between CMT and abdominal venous congestion caused by left renal vein compression, or ‘nutcracker phenomenon,’ has not been previously reported. Case Report: A 91-year-old woman, who died from a cerebrovascular accident (CVA), underwent a cadaveric examination at our medical school. On examination of the abdomen, there was an incidental finding of CMT. The arterial and venous diameters were measured, and vascular histopathology was undertaken. The vascular anatomy was consistent with CMT type 1-b. Nutcracker phenomenon (NCP) (left renal vein compression) was seen anatomically as dilatation and engorgement of the left renal vein, relative to the right renal vein (10.77±0.13 mm vs. 4.49±0.56 mm, respectively), and dilatation and engorgement of the left ovarian vein, relative to the right ovarian vein (4.37±0.15 mm vs. 1.06±0.09 mm, respectively) with left ovarian varicocele. The aortoceliac angle (ACA) and the aortomesenteric angle (AMA) approached zero degrees. Conclusions: We have described a rare anatomic finding of CMT that created an acute AMA and NCP. Awareness of this rare association between CMT and NCP by clinicians, vascular surgeons, and radiologists may be of value in the future evaluation and surgical management of patients who present clinically with ‘nutcracker syndrome.’