Squalamine inhibits angiogenesis and solid tumor growth in vivo and perturbs embryonic vasculature.

The novel aminosterol, squalamine, inhibits angiogenesis and tumor growth in multiple animal models. This effect is mediated, at least in part, by blocking mitogen-induced proliferation and migration of endothelial cells, thus preventing neovascularization of the tumor. Squalamine has no observable effect on unstimulated endothelial cells, is not directly cytotoxic to tumor cells, does not alter mitogen production by tumor cells, and has no obvious effects on the growth of newborn vertebrates. Squalamine was also found to have remarkable effects on the primitive vascular bed of the chick chorioallantoic membrane, which has striking similarities to tumor capillaries. Squalamine may thus be well suited for treatment of tumors and other diseases characterized by neovascularization in humans.

Expansile skeletal hyperphosphatasia: a new familial metabolic bone disease.

We describe a new familial metabolic bone disease characterized by expanding hyperostotic long bones, early onset deafness, premature tooth loss, and episodic hypercalcemia. The condition affects a mother and daughter studied at the age of 36 years and 11 years, respectively. Both individuals lost all hearing in early childhood and suffered premature shedding of teeth. Skeletal pains began just before puberty. Swelling and aching of most middle phalanges in the hands is an especially troublesome manifestation. The mother also had episodes of symptomatic hypercalcemia first documented in late childhood and subsequently during intercurrent illness and postpartum lactation. Radiographs show hyperostosis and/or osteosclerosis predominantly in the skull and appendicular skeleton. Long bones also are expanded considerably, especially the middle phalanges in the fingers. The mother's skeletal abnormalities are more severe. Biochemical parameters of bone turnover, including serum alkaline phosphatase (ALP) activity, are elevated substantially. In the proposita, dynamic histomorphometry of nondecalcified sections of iliac crest revealed rapid skeletal remodeling. In the mother, who had been treated with bisphosphonates, electron microscopy (EM) showed disorganized collagen bundles as well as necrotic and apoptotic bone cells but no osteocytic osteolysis. Measles virus gene transcripts were not detected in peripheral blood monocytes. Karyotyping was normal, 46,XX. Hyperphosphatasia with bone disease previously has been reported as either a sporadic or autosomal recessive condition. Expansile skeletal hyperphosphatasia (ESH) is probably inherited as an autosomal dominant trait with a high degree of penetrance.

GNAS1 mutation and Cbfa1 misexpression in a child with severe congenital platelike osteoma cutis.

We evaluated a 7-year-old girl with severe platelike osteoma cutis (POC), a variant of progressive osseous heteroplasia (POH). The child had congenital heterotopic ossification of dermis and subcutaneous fat that progressed to involve deep skeletal muscles of the face, scalp, and eyes. Although involvement of skeletal muscle is a prominent feature of POH, heterotopic ossification has not been observed in the head, face, or extraocular muscles. The cutaneous ossification in this patient was suggestive of Albright hereditary osteodystrophy (AHO); however, none of the other characteristic features of AHO were expressed. Inactivating mutations of the GNAS1 gene, which encodes the alpha-subunit of the stimulatory G protein of adenylyl cyclase, is the cause of AHO. Mutational analysis of GNAS1 using genomic DNA of peripheral blood and of lesional and nonlesional tissue from our patient revealed a heterozygous 4-base pair (bp) deletion in exon 7, identical to mutations that have been found in some AHO patients. This 4-bp deletion in GNAS1 predicts a protein reading frameshift leading to 13 incorrect amino acids followed by a premature stop codon. To investigate pathways of osteogenesis by which GNAS1 may mediate its effects, we examined the expression of the obligate osteogenic transcription factor Cbfa1/RUNX2 in lesional and uninvolved dermal fibroblasts from our patient and discovered expression of bone-specific Cbfa1 messenger RNA (mRNA) in both cell types. These findings document severe heterotopic ossification in the absence of AHO features caused by an inactivating GNAS1 mutation and establish the GNAS1 gene as the leading candidate gene for POH.

Mast cell involvement in fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a catastrophic genetic disorder of progressive heterotopic ossification associated with dysregulated production of bone morphogenetic protein 4 (BMP4), a potent osteogenic morphogen. Postnatal heterotopic ossification in FOP is often heralded by hectic episodes of severe post-traumatic connective tissue swelling and intramuscular edema, followed by an intense and highly angiogenic fibroproliferative mass. The abrupt appearance, intense size, and rapid intrafascial spread of the edematous preosseous fibroproliferative lesions implicate a dysregulated wound response mechanism and suggest that cells and mediators involved in inflammation and tissue repair may be conscripted in the growth and progression of FOP lesions. The central and coordinate role of inflammatory mast cells and their mediators in tissue edema, wound repair, fibrogenesis, angiogenesis, and tumor invasion prompted us to investigate the potential involvement of mast cells in the pathology of FOP lesions. We show that inflammatory mast cells are present at every stage of the development of FOP lesions and are most pronounced at the highly vascular fibroproliferative stage. Mast cell density at the periphery of FOP lesional tissue is 40- to 150-fold greater than in normal control skeletal muscle or in uninvolved skeletal muscle from FOP patients and 10- to 40-fold greater than in any other inflammatory myopathy examined. These findings document mobilization and activation of inflammatory mast cells in the pathology of FOP lesions and provide a novel and previously unrecognized target for pharmacologic intervention in this extremely disabling disease.

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.

Lepidic intrapulmonary growth of malignant mesothelioma presenting as recurrent hydropneumothorax.

We report a case of malignant mesothelioma with unusual clinical and histological findings. The patient presented with recurrent hydropneumothorax and minimal pleural thickening on chest computed tomography (CT). Histologically, the pleura was involved by the malignant mesothelioma, albeit to a limited degree. Unexpectedly, the lung parenchyma from two different lobes showed focal nests of mesothelioma cells filling the alveolar spaces and growing on the luminal surface of the alveolar septa, closely resembling the multicentric growth pattern of bronchioloalveolar adenocarcinoma. Immunohistochemical and ultrastructural studies confirmed that the pulmonary lesions were an extension of the malignant mesothelioma. This case illustrates clinically, the importance of a high index of suspicion for malignancy in older patients with unexplained recurrent hydropneumothorax; and histologically the potential of malignant mesothelioma to invade the lung at an early stage of growth.

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.

Bone involvement in sinusitis: an apparent pathway for the spread of disease.

OBJECTIVES/HYPOTHESIS: To study the effects of bone involvement in experimentally induced sinusitis and the effect of involved bone on the overlying mucosa. STUDY DESIGN: Animal study. METHODS: Sinusitis was induced unilaterally with Pseudomonas aeruginosa in the maxillary sinus of 19 New Zealand white rabbits. At 6 weeks, the pathogenic organism was confirmed by culture, and a segment of the bone from the medial wall of the sinus implanted in a submucosal pocket in the opposite sinus. The rabbits were killed at predetermined time intervals up to 13 weeks from sinusitis induction, and en bloc sinus sections were decalcified and stained. RESULTS: The implanted bone reabsorbed partially or totally in all specimens. However, the study revealed clear histological evidence of bone involvement adjacent to the infected sinuses and the bony changes extended to the noninfected side in all specimens. The histological findings were identical to those seen in chronic osteomyelitis. CONCLUSIONS: This study demonstrates the ability for pseudomonal sinusitis, at least in the presence of surgical intervention, to involve bone at a distance from the site of primary infection in the absence of intervening mucosal disease. If confirmed with additional organisms and models, these findings have significant implications for the therapeutic management of chronic sinus disease.

Histology and histomorphometry of ethmoid bone in chronic rhinosinusitis.

Mucosal changes have been well described in chronic sinusitis, yet little is known about the underlying bone, despite clinical and experimental evidence suggesting that bone may be involved in chronic sinusitis. Techniques of undecalcified bone analysis were used for detailed histologic examination of ethmoid bone in chronic sinusitis compared with controls. Bone synthesis, resorption, and inflammatory cell presence were specifically assessed. Additionally, histomorphometry techniques were used to determine ethmoid bone physiology in individuals undergoing surgery for chronic sinusitis. Overall, individuals undergoing surgery for chronic sinusitis were found to have evidence of marked acceleration in bone physiology with histologic changes including new bone formation, fibrosis, and presence of inflammatory cells. These findings are compared with osteomyelitis in long bone and the jaw. The suggestion that underlying bone may serve as a catalyst for chronic sinusitis is supported and implications for therapy are discussed.

Identification and characterization of calcifying valve cells from human and canine aortic valves.

BACKGROUND AND AIM OF THE STUDY: Cardiac valve calcification is the predominant pathology in patients needing valve replacement. The aim of this study was to determine if aortic valve cells calcify spontaneously and, if so, to characterize the nodular complex and response to growth factors. METHODS: Aortic valves were obtained from humans undergoing surgical valve replacement, and from female dogs. The valvular endothelium was removed and explants cultured in medium. RESULTS: A population of valvular interstitial cells spontaneously formed distinct calcified nodules containing hydroxyapatite within two to three weeks in canine and within six weeks in human aortic valves. The nodules contained an inner ring of dead cells surrounded by an outer ring of living cells. Cells associated with nodules had osteoblast-like characteristics and stained positively for extracellular bone matrix proteins. Incubating canine cells with potential calcifying stimuli tested the stimulus for calcification. The rate of nodule formation was increased with transforming growth factor beta-1 (+25 nodules), 25-hydroxycholesterol (+9 nodules) and bone morphogenetic protein 2 (+4 nodules) as compared with vehicle control (+3 nodules) over 25 days. CONCLUSIONS: We identified a population of valvular interstitial cells with osteoblast-like characteristics that spontaneously form calcific nodules in cell culture. In addition, the rate of calcific nodule formation was increased with transforming growth factor beta-1 and 25-hydroxycholesterol. Further study of these 'calcifying valve cells' may yield a new in vitro model for testing therapy aimed at preventing calcific valve stenosis.

Properties of coralline hydroxyapatite and expanded polytetrafluoroethylene membrane in the immature craniofacial skeleton.

Although extensive research regarding the treatment of calvarial defects has been done in adult models, little is known about the response in the maturing skeleton. The role of coralline hydroxyapatite and expanded polytetrafluoroethylene membrane in augmenting bone growth and repair of calvarial defects in a neonatal model is explored. Utilizing a 3-week-old neonatal swine model, bone growth into 28 calvarial defects was measured. After exposure of the calvaria in seven animals, four defects of 10 mm in diameter were created. In each animal, one defect was treated with a 10-mm disc of porous hydroxyapatite alone (Interpore 500, Interpore International), and a second defect was covered with an expanded polytetrafluoroethylene membrane (Gore-Tex OV-6) secured by four 3-mm microscrews (Luhr Microsystem, Howe-Medica Inc.). The third defect combined an implanted hydroxyapatite disc covered by an expanded polytetrafluoroethylene membrane, whereas the fourth defect served as an untreated control. Histology and histomorphometry were performed on undecalcified specimens harvested at 6 weeks after surgery. In both hydroxyapatite groups, the bone growth into the inorganic matrix provided complete osseous union in all specimens, and the amount of fibrosis was significantly lower (p < 0.02) in comparison with the control. Unexpectedly, there was significant osteoclastic resorption of the hydroxyapatite matrix (35.1 percent decrease) with simultaneous bone deposition and remodeling. The addition of an expanded polytetrafluoroethylene membrane covering the hydroxyapatite implant provided an insignificant advantage in bone growth (27.3 percent versus 28.3 percent, respectively). Finally, the expanded polytetrafluoroethylene membrane alone afforded no qualitative advantage secondary to intrusion of brain and dura into the defect as well as displacement of the membrane inward during appositional growth, leading to incomplete healing of the defect with thinning of the surrounding cranial bone. Unique in this maturing model was morphologic evidence of complete union at the calvaria-hydroxyapatite interface in all specimens as well as active remodeling of the hydroxyapatite matrix. The results of this study suggest that porous hydroxyapatite may be a suitable bone substitute in maturing calvarial bone defects, achieving superior osseous integration and volumetric bone gain while undergoing concurrent resorption and remodeling.

Management and prevention of vitamin D deficiency rickets in captive-born juvenile chimpanzees (Pan troglodytes).

Vitamin D deficiency rickets was diagnosed in three juvenile chimpanzees (Pan troglodytes) raised indoors under skylights and consuming only breast milk. Two cases detected early had mild but characteristic radiographic changes. More advanced disease presented with florid x-ray features of rickets and pathologic fractures, as well as hypocalcemia, hypophosphatemia, and low serum 25-hydroxyvitamin D levels. Treatment by a single injection of vitamin D2 in sesame oil (slow release) followed by daily oral supplementation with vitamin D2 corrected the condition. On the basis of experience with these cases and comparison with rickets in humans, a prevention protocol for mother-reared, inside-housed, chimpanzee juveniles was developed. Injection with slow release vitamin D2 (5,000 IU i.m. once) at 4 mo of age, followed by oral supplementation of 400 IU vitamin D2 daily until weaning, prevents rickets in juvenile chimpanzees raised indoors.

Histomorphometry of the aging human patella: histologic criteria and controls.

OBJECTIVE: A histomorphometric analysis of patellae from necropsies on persons between the third and tenth decades of life was carried out to trace the natural history of osteoarthritis. DESIGN: Minutiae of the histological changes in the surface and basilar portions of the articular cartilage were developed as criteria for the quantitation. A total of 99 patellas were harvested in the stated age range. The present study reports the results of ten grossly and radiologically normal specimens from subjects 23-32 years old served as controls. RESULTS: None of the control patellae were entirely histologically normal. Abnormality of the cartilage surface did not consistently proceed remodeling at the attachment to the subchondral plate. CONCLUSIONS: This observation throws into question the concept that osteoarthritis has a single histogenesis or always arises in articular cartilage.

Osteogenic induction in hereditary disorders of heterotopic ossification.

The formation of heterotopic bone within soft connective tissue is a common feature of at least three distinct genetic disorders of osteogenesis in humans: fibrodysplasia ossificans progressiva; progressive osseous heteroplasia; and Albright hereditary osteodystrophy. The pathobiologic characteristics of osteogenic induction, the histopathologic features of osteogenesis, the anatomic distribution of heterotopic lesions, and the developmental patterns of disease progression differ among all three conditions. The molecular and cellular basis of redirecting a mature connective tissue phenotype to form bone is a remarkable biological phenomenon with enormous implications for the control of bone regeneration, fracture healing, and disorders of osteogenesis.

Fibrodysplasia ossificans progressiva why do some people have two skeletons?

Fibrodysplasia ossificans progressiva (FOP) is one of the rarest genetic conditions known. People who have this disorder essentially form two skeletons: a normal one during embryogenesis and a heterotopic one after birth. Although the general phenotype of the disease, including the presence of congenital malformations of the great toes, is constant among individuals, there is wide variation in the severity of the disorder. Studies to identify the cause of FOP currently are focused on a candidate gene approach. Bone morphogenetic proteins (BMPs) are bone-inducing morphogens that are involved in the developmental organization of the skeleton and are excellent candidate genes for disorders of heterotopic osteogenesis. We recently have demonstrated that overexpression of BMP 4 in cells from patients who have FOP is associated with this disorder. We are continuing to investigate the role of BMP-4 in the pathophysiology of FOP. An analysis of the genetic and cellular pathways that are activated ectopically in patients who have FOP will help elucidate how bone forms and grows and will lead to more effective treatments of orthotopic and heterotopic osteogenesis.

Chordomalike soft tissue sarcoma in the leg: a light and electron microscopic and immunohistochemical study.

A soft tissue tumor in the leg of a 67-year-old woman is described. This large tumor below the knee area infiltrated extensively the deep and superficial soft tissues but did not involve the bones. The tumor cells formed nodules resembling the architecture seen in chondroid tumors and chordoma. The tumor cells were often vacuolized, and there was extracellular myxoid matrix similar to that in chordoma or myxoid chondrosarcoma. Immunohistochemistry showed keratins 8 and 19, epithelial membrane antigen, and vimentin in most tumor cells, and there was also S-100 protein positivity in a number of tumor cells. Electron microscopy showed desmosomelike cell junctions and bundles of intermediate filaments resembling those seen in many epithelial neoplasms. Thus the tumor resembled chordoma in many respects. Because clinically no other primary tumor was found, this tumor is probably a chordomalike primary soft tissue sarcoma different from typical extraskeletal myxoid chondrosarcoma or chordoid sarcoma.

Primary chondrosarcoma of the head and neck in pediatric patients: a clinicopathologic study of 14 cases with a review of the literature.

BACKGROUND: Primary chondrosarcoma of the head and neck in the pediatric age group is rare. The literature contains several single cases and small series; however, to the authors' knowledge, there has been no previous comprehensive larger study to evaluate the clinicopathologic aspects of these tumors. METHODS: Fourteen cases of chondrosarcoma of the head and neck from patients age 18 years or younger, diagnosed between 1970 and 1997, were retrieved from the Otorhinolaryngic-Head & Neck Tumor Registry of the Armed Forces Institute of Pathology. No secondary sarcomas (radiation-induced or arising in association with Maffucci syndrome or Ollier disease) were included. Clinical, radiographic, and histologic features were reviewed and patient follow-up obtained. RESULTS: The patients included 6 girls and 8 boys ages 3-18 years (mean, 11.8 years). Patient symptoms (nasal stuffiness or discharge, sinusitis, headaches, or a mass lesion) were related to tumor location and were present for an average of 7.2 months. No genetic abnormalities were documented. The tumors most frequently involved the maxillary sinus (n=4), followed by the mandible (n=3), nasal cavity (n=2), and neck (n=2), with 1 each of the nasopharynx, orbit, and base of the skull. The tumors ranged in size from 2.0 to 15.0 cm (mean, 3.1 cm). All tumors were invasive and malignant as determined by radiology and/or histology. The tumors were Grade 1 (n=9), Grade 2 (n=1), or Grade 3 (mesenchymal, n=2; dedifferentiated n=2). All patients were treated by surgery, followed by radiation (n=5) and/or chemotherapy (n=2). Follow-up was available for 11 patients; all were alive (at a mean of 14.8 years), with only a single patient demonstrating evidence of residual/ recurrent tumor (at 16.6 years). CONCLUSIONS: Primary head and neck chondrosarcoma in the pediatric population is typically low grade and occurs in the maxillary sinus or mandible. Despite the invasive and high grade nature of some of these tumors, there is an excellent long term prognosis for patients in this age group with tumors in these locations.

Bull's-eyes and halos: useful MR discriminators of osseous metastases.

To evaluate the presence of (a) a focus of high signal intensity in the center of an osseous lesion (bull's-eye) as a negative discriminator for metastasis and (b) a rim of high signal intensity around an osseous lesion (halo) as a positive discriminator, a retrospective study was performed in 47 patients with osseous lesions suspect for metastatic disease who underwent magnetic resonance (MR) imaging of the pelvis. The findings in 17 patients with proved osseous metastasis were compared with those in 30 patients not believed to have metastatic disease; T1- and T2-weighted MR images were evaluated. The bull's-eye sign was found to be a specific indicator of normal hematopoietic marrow (sensitivity, 95%; specificity, 99.5%). The halo sign and diffuse signal hyperintensity were a strong indicator of metastatic disease (sensitivity, 75%; specificity, 99.5%). These results suggest that use of the bull's-eye sign as a discriminator of benign disease and use of the halo sign as a discriminator of metastasis help characterize suspect areas of marrow lesions.

Giant cell tumor of the larynx: a clinicopathologic series of eight cases and a review of the literature.

True giant cell tumors of the larynx (GCTL) are quite rare, and only individual case reports are documented in the literature. Eight cases of GCTL were identified in the Otorhinolaryngic Pathology Tumor Registry between 1966 and 2000. There were 2 women and 6 men, ages 26 to 62 years (mean, 44.5 yrs). Patients presented with a palpable neck mass (n = 5), airway obstruction (n = 3), hoarseness (n = 3), and dysphagia (n = 2). All tumors involved the thyroid cartilage, a few with local extension. The mean tumor size was 4.1 cm. Histologically, the tumors showed no connection to the surface epithelium and arose in sites of ossification. The tumors had an expansile, infiltrative growth and consisted of numerous multinucleated osteoclast-like giant cells within a cellular stroma composed of plump, oval mononuclear cells. Of interest was that the nuclei of the giant cells were similar to the nuclei of the stromal cells. Treatment included biopsy only with adjuvant therapy (n = 2), local resection (n = 3), and total laryngectomy (n = 3). Follow-up showed 5 patients were alive without evidence of disease (mean follow-up, 6.9 yrs); 2 died of unrelated causes (mean survival, 22.2 yrs). No patients developed recurrences. GCTL are rare tumors that can cause significant airway obstruction. Complete surgical resection yields an excellent outcome without adjuvant therapy.