Francis H. Gannon

Bone Formation and Inflammation in Cardiac Valves

BackgroundFor nearly a century, the mechanical failure of calcified heart valves was attributed to a passive degenerative process. Recently, several case reports described bone formation in surgically excised heart valves and suggested an unexpected process of tissue repair. Methods and ResultsWe studied the prevalence and pathology of heterotopic ossification in 347 surgically excised heart valves (256 aortic, 91 mitral) in 324 consecutive patients (182 men, 142 women; mean age 68 years) who underwent cardiac valve replacement surgery between 1994 and 1998. The valves were examined microscopically to determine the prevalence and features of bone formation and remodeling. Two hundred eighty-eight valves (83%) had dystrophic calcification. Mature lamellar bone with hematopoietic elements and active bone remodeling were present in 36 valves (13%) with dystrophic calcification. Endochondral bone formation, similar to that seen in normal fracture repair, was identified in 4 valves. Microfractures were present in 92% of all valves with ossification. Neoangiogenesis was found in all valves with ossification. Bone morphogenetic proteins 2 and 4 (BMP 2/4), potent osteogenic morphogens, were expressed by myofibroblasts and preosteoblasts in areas adjacent to B- and T-lymphocyte infiltration in valves where ossification was identified. Mast cells were present in calcified and ossified valves and were especially prominent in atheromatous regions. ConclusionsHeterotopic ossification consisting of mature lamellar bone formation and active bone remodeling is a relatively common and unexpected finding in end-stage valvular heart disease and may be associated with repair of pathological microfractures in calcified cardiac valves.

OVEREXPRESSION OF AN OSTEOGENIC MORPHOGEN IN FIBRODYSPLASIA OSSIFICANS PROGRESSIVA

BACKGROUND Fibrodysplasia ossificans progressiva is a heritable disorder of connective tissue characterized by congenital malformation of the great toes and postnatal formation of ectopic bone. Although the disorder was first described more than 300 years ago, the genetic defect and pathophysiology remain unknown. Bone morphogenetic proteins are potent bone-inducing morphogens that participate in the developmental organization of the skeleton, and increased production of one or more of these proteins has been proposed as the cause of fibrodysplasia ossificans progressiva. METHODS We studied lymphoblastoid cell lines established from peripheral-blood mononuclear cells of patients with fibrodysplasia ossificans progressiva and fibroblast-like cell lines derived from lesional and nonlesional tissue. We used Northern blot analysis and ribonuclease protection assays to measure the expression of messenger RNA (mRNA) of bone morphogenetic proteins 1 to 7 and immunohistochemical analysis to examine protein expression. RESULTS Among the bone morphogenetic proteins and mRNAs examined, only bone morphogenetic protein 4 and its mRNA were present in increased levels in cells derived from an early fibroproliferative lesion in a patient with fibrodysplasia ossificans progressiva. Bone morphogenetic protein 4 mRNA was expressed in lymphoblastoid cell lines from 26 of 32 patients with fibrodysplasia ossificans progressiva but from only 1 of 12 normal subjects (P<0.001). Bone morphogenetic protein 4 and its mRNA were detected in the lymphoblastoid cell lines from a man with fibrodysplasia ossificans progressiva and his three affected children (two girls and a boy), but not from the childrens unaffected mother. No other bone morphogenetic proteins were detected. CONCLUSIONS Overexpression of a potent bone-inducing morphogen (bone morphogenetic protein 4) in lymphocytes is associated with the disabling ectopic osteogenesis of fibrodysplasia ossificans progressiva.

The histopathology of fibrodysplasia ossificans progressiva. An endochondral process.

In order to better characterize the biological features of fibrodysplasia ossificans progressiva, we reviewed the histopathological specimens from eleven patients (twelve biopsies) who had a confirmed diagnosis of the disease. All of the biopsies had been performed in children, to exclude the diagnosis of a malignant lesion. In no instance was the diagnosis of fibrodysplasia ossificans progressiva considered before the biopsy. The results of a lesional biopsy in all eleven patients revealed normal endochondral osteogenesis at heterotopic sites. The results of biopsy of an early lesion in six children were misinterpreted as revealing a diagnosis of fibromatosis or sarcoma before the roentgenographic appearance of ossification. Immunohistochemical studies of sections of the earliest lesion demonstrated S-100 antigen positivity before the histological appearance of differentiated osteochondral tissue. The presence of congenital malformation of the great toes and of postnatal heterotopic endochondral osteogenesis strongly suggests that fibrodysplasia ossificans progressiva is a disorder of defective induction of endochondral osteogenesis. This study established the predominant histopathological findings associated with fibrodysplasia ossificans progressiva and can serve as a basis for postulation of a candidate gene in the pathogenesis of the disorder. A lesional biopsy is not needed to make the diagnosis; biopsy uniformly exacerbates the condition and should be avoided.

Chondrosarcoma of the larynx: a clinicopathologic study of 111 cases with a review of the literature.

Chondrosarcomas of the larynx are rare tumors accounting for about 0.5% of all laryngeal primary tumors. A total of 111 laryngeal chondrosarcoma cases, diagnosed between 1970 and 1997, were retrieved from the Otorhinolaryngic-Head & Neck Tumor Registry of the Armed Forces Institute of Pathology. There was a 3.6:1 male/female ratio of patients 25–91 years of age (mean, 64.4 years). Patients presented most frequently with hoarseness (n = 72 patients) present for a mean of 28.2 months. The majority of tumors involved the cricoid cartilage (n = 77) with a mean size of 3.5 cm. All tumors were invasive and malignant by radiology and/or histology (into bone within the ossified laryngeal cartilages in 52 tumors). Most tumors were low-grade lesions: grade 1 (n = 51), grade 2 (n = 54); there were six grade 3 tumors. An associated benign chondroma with (n = 41 tumors) or without ischemia (n = 24 tumors) was noted. All patients had surgery and five had radiation therapy. Wide excision or voice-sparing surgery was used in 73 patients, whereas 37 patients had a laryngectomy. Recurrences occurred in 20 (18%) patients, 10 of whom underwent salvage laryngectomy. At the last follow-up, 102 patients had no evidence of disease (alive or dead, mean 11.2 years) and five patients had evidence of disease (alive, one patient, 6.5 years; dead, four patients, mean 6.4 years). The six patients with high-grade chondrosarcoma were all without disease at the last follow-up (mean, 15.1 years). There was no difference in clinical outcome based on grade (p = 0.210), location (p = 0.078), or treatment (p = 0.607) but was worse for patients with a myxoid-type chondrosarcoma (p = 0.044). Primary laryngeal chondrosarcomas are typically low- to moderate-grade lesions involving the cricoid cartilage, frequently associated with a chondroma. They usually portend an excellent overall long-term prognosis with initial conservative voice-sparing surgery.

Bone morphogenetic protein 2 4 in early fibromatous lesions of fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder characterized by congenital malformation of the great toes and progressive heterotopic ossification in distinct anatomic patterns. Early preosseous lesions in FOP are clinically and histologically indistinguishable from the lesions of aggressive juvenile fibromatosis (AJF). Although the genetic defect in FOP is unknown, bone morphogenetic proteins (BMPs) 2 and 4 are plausible candidates genes. To determine if there is a difference in BMP 2/4 expression in the early fibromatous lesions of the two conditions, we performed immunohistochemical studies with a monoclonal antibody to BMP 2/4 on the earliest detectable fibromatous lesions of FOP and compared them with histologically identical lesions resected from children who had AJF. Fibromatous cells from the early FOP lesions exhibited immunostaining for BMP 2/4, whereas histologically indistinguishable fibromatous cells from AJF lesions showed no evidence of BMP 2/4 immunostaining. It is incumbent on all physicians who treat patients with suspected fibromatosis to examine the toes to rule out FOP and to avoid unnecessary diagnostic biopsies because surgical trauma induces further bone formation in patients who have FOP. However, if diagnostic confusion still exists and a biopsy is performed, immunostaining with BMP 2/4 antibody may resolve the diagnostic dilemma between FOP and AJF before the appearance of heterotopic ossification is observed in the FOP lesions. Our data suggest that the BMP 2/4 subfamily of secreted proteins may be involved in the pathogenesis of the FOP lesions.

Progressive osseous heteroplasia: a distinct developmental disorder of heterotopic ossification. Two new case reports and follow-up of three previously reported cases.

In vestigation J)erforlned at the Department of Orthopaedic Surge, , the University of Pennsylvania School of Medicine, Philadelphia: the Departmeius of Pathology and Orthopaedic Surgery. Louisiana State University, New Orleans; the Department of Patbzology, the Jefferson University School of Medicine, Philadelphia: the Department of Paediatrics and Child Health, University of Otago, Dunedin, and the Division of Human Genetics and Molecular Biology, Children ‘s Hospital of Philadelphia, Philadelphia

Acute lymphocytic infiltration in an extremely early lesion of fibrodysplasia ossificans progressiva.

A 2-year-old child with fibrodysplasia ossificans progressiva underwent a muscle biopsy of a very early lesion, and had findings that showed the earliest stage ever seen in the histopathology of fibrodysplasia ossificans progressiva. This very early stage consisted of intense perivascular lymphocytic infiltration into normal appearing skeletal muscle. A nearly identical histopathologic sequence was noted in a cat with phenotypic features similar to those of fibrodysplasia ossificans progressiva in humans. These new findings represent the earliest documented changes that have ever been noted in fibrodysplasia ossificans progressiva, and provide further histopathologic support for the recent discovery that lymphocytes may play a role in the pathogenesis of heterotopic ossification in fibrodysplasia ossificans progressiva.

Sensory nerve induced inflammation contributes to heterotopic ossification

Heterotopic ossification (HO), or bone formation in soft tissues, is often the result of traumatic injury. Much evidence has linked the release of BMPs (bone morphogenetic proteins) upon injury to this process. HO was once thought to be a rare occurrence, but recent statistics from the military suggest that as many as 60% of traumatic injuries, resulting from bomb blasts, have associated HO. In this study, we attempt to define the role of peripheral nerves in this process. Since BMP2 has been shown previously to induce release of the neuroinflammatory molecules, substance P (SP) and calcitonin gene related peptide (CGRP), from peripheral, sensory neurons, we examined this process in vivo. SP and CGRP are rapidly expressed upon delivery of BMP2 and remain elevated throughout bone formation. In animals lacking functional sensory neurons (TRPV1−/−), BMP2‐mediated increases in SP and CGRP were suppressed as compared to the normal animals, and HO was dramatically inhibited in these deficient mice, suggesting that neuroinflammation plays a functional role. Mast cells, known to be recruited by SP and CGRP, were elevated after BMP2 induction. These mast cells were localized to the nerve structures and underwent degranulation. When degranulation was inhibited using cromolyn, HO was again reduced significantly. Immunohistochemical analysis revealed nerves expressing the stem cell markers nanog and Klf4, as well as the osteoblast marker osterix, after BMP2 induction, in mice treated with cromolyn. The data collectively suggest that BMP2 can act directly on sensory neurons to induce neurogenic inflammation, resulting in nerve remodeling and the migration/release of osteogenic and other stem cells from the nerve. Further, blocking this process significantly reduces HO, suggesting that the stem cell population contributes to bone formation. J. Cell. Biochem. 112: 2748–2758, 2011.

Use of a Chimeric Adenovirus Vector Enhances BMP2 Production and Bone Formation

Recombinant adenoviral vectors have potential for the treatment of a variety of musculoskeletal defects and such gene therapy systems have been a recent research focus in orthopedic surgery. In studies reported here, two different adenovirus vectors have been compared for their ability to transduce human bone marrow mesenchymal stem cells (hBM-MSCs) and elicit bone formation in vivo. Vectors consisted either of standard adenovirus type 5 (Ad5) vector or a chimeric adenovirus type 5 vector that contains an adenovirus type 35 fiber (Ad5F35), which has been recently demonstrated to bestow a different cellular tropism, and a complete cDNA encoding human bone morphogenetic 2 (BMP2). Studies were also conducted to compare the transduction efficiency of these vectors using enhanced green fluorescent protein (GFP). hBM-MSCs transduced with Ad5F35 vectors had higher levels of transgene expression than those transduced with Ad5 vectors. The results also demonstrate that hBM-MSCs lack the coxsackie-adenovirus receptor (CAR), which is responsible for cellular adsorption of Ad5. Therefore, the data suggest that Ad5 virus adsorption to hBM-MSCs is inefficient. Ad5BMP2- or Ad5F35BMP2-transduced hBM-MSCs were also compared in an in vivo heterotopic bone formation assay. Mineralized bone was radiologically identified only in muscle that received the Ad5F35BMP2 transduced hBM-MSCs. In summary, Ad5F35BMP2 can efficiently transduce hBM-MSCs leading to enhanced bone formation in vivo.

Proteoglycan 4 expression protects against the development of osteoarthritis.

Proteoglycan 4 protects from age-related and posttraumatic osteoarthritis development. Rubbing Arthritis the Wrong Way There are two main forms of joint inflammation (arthritis). Patients with rheumatoid arthritis develop joint inflammation because of an autoimmune reaction, whereas wear and tear is thought to contribute to the development of osteoarthritis. Indeed, most people have at least some symptoms of osteoarthritis by the age of 70. Treatment is mostly limited to reducing symptoms. Now, Ruan et al. provide a new mechanism for a potential new therapy for osteoarthritis. Proteoglycan 4 (lubrican, PRG4) has been previously thought to protect against osteoarthritis by acting as a lubricant between bones in a joint. Here, the authors show that not only does PRG4 protect against the development of osteoarthritis in both aging and trauma mouse models, but that its effect may extend beyond that of a mere lubricant. Indeed, PRG4 has a direct chondroprotective effect in these models, suggesting that it may form the basis for a new disease-altering therapy for osteoarthritis. Osteoarthritis (OA) is a common degenerative condition that afflicts more than 70% of the population between 55 and 77 years of age. Although its prevalence is rising globally with aging of the population, current therapy is limited to symptomatic relief and, in severe cases, joint replacement surgery. We report that intra-articular expression of proteoglycan 4 (Prg4) in mice protects against development of OA. Long-term Prg4 expression under the type II collagen promoter (Col2a1) does not adversely affect skeletal development but protects from developing signs of age-related OA. The protective effect is also shown in a model of posttraumatic OA created by cruciate ligament transection. Moreover, intra-articular injection of helper-dependent adenoviral vector expressing Prg4 protected against the development of posttraumatic OA when administered either before or after injury. Gene expression profiling of mouse articular cartilage and in vitro cell studies show that Prg4 expression inhibits the transcriptional programs that promote cartilage catabolism and hypertrophy through the up-regulation of hypoxia-inducible factor 3α. Analyses of available human OA data sets are consistent with the predictions of this model. Hence, our data provide insight into the mechanisms for OA development and offer a potential chondroprotective approach to its treatment.