Jason A. Horton

Cross-sectional study of bone mineral density in adult survivors of solid pediatric cancers.

To investigate the hypothesis that survivors of pediatric solid cancer have low bone mineral density, a cross-sectional study was done of subjects who had received treatment for pediatric solid tumors before 16 years of age and were less than 40 years old at follow-up. Excluded were subjects treated for acute lymphoblastic leukemia or those who had received cranial irradiation, total body radiation, or nonautologous bone marrow transplant. The study group consisted of 38 subjects, with the most common diagnoses being lymphoma (n = 17), sarcoma (n = 8), Wilms tumor (n = 5), and neuroblastoma (n = 4). Median age was 22 years (range 12-32). Time from diagnosis of underlying cancer averaged 12.6 years (range 5.5-20.3). Using criteria of osteopenia (Z-score ≤−1.0 and >−2.0) and osteoporosis (Z-score ≤ −2.0) for any one or more areas including total body, lumbar spine, total hip, or femoral neck density, 13 of the 38 subjects (34%) had osteopenia or osteoporosis. A further six subjects (16%) had isolated upper extremity osteopenia or osteoporosis. Multivariate analysis showed a direct relationship between the number of chemotherapy drugs administered and the presence of osteopenia or osteoporosis in the lower extremities (P = 0.03). Young survivors of childhood solid tumors are at increased risk of developing premature osteopenia or osteoporosis, and screening evaluations and follow-up are warranted.

Ontogeny of Skeletal Maturation in the Juvenile Rat

Systemic regulation of the cellular processes that produce endochondral elongation and endochondral mineralization during postnatal skeletal maturation are not completely understood. In particular, a mechanism coupling the decline of cellular activity in the bone microenvironment to the onset of sexual maturity remains elusive. The purpose of this study was to empirically integrate the dynamic progression of bone mineral accrual and endochondral elongation as a function of animal age in growing male and female Sprague‐Dawley rats. We used serial dual‐energy X‐ray absorptiometry (DXA) and radiography to study the temporal progression of bone growth and mineral accrual from weaning to adulthood. We observed that skeletal maturation proceeds in a pattern adequately described by the Gompertz function. During this period of growth, we found that serum markers of osteoblastic bone formation declined with age, while osteoclastic bone resorption activity remained unchanged. We also report a slight lag in the age at inflection in the rate of bone mineral accrual relative to the rate of tibial elongation and that both endochondral processes eventually come to asymptotic equilibrium by approximately 20 weeks of age. In addition, we studied tibial growth plate histomorphometry at select time points through 1 year of age. We report that, despite the histologic persistence of physeal cartilage, very little proliferative or elongative activity was measured in this tissue beyond 20 weeks of age. Taken together, these data provide insight to the temporal coordination of postnatal endochondral growth processes. Anat Rec, 291:283–292, 2008.

Microarray analysis of perichondral and reserve growth plate zones identifies differential gene expressions and signal pathways.

In the growth plate, the reserve and perichondral zones have been hypothesized to have similar functions, but their exact functions are poorly understood. Our hypothesis was that significant differential gene expression exists between perichondral and reserve chondrocytes that may differentiate the respective functions of these two zones. Normal Sprague-Dawley rat growth plate chondrocytes from the perichondral zone (PC) and reserve zone (RZ) were isolated by laser microdissection and then subjected to microarray analysis. In order to most comprehensively capture the unique features of the two zones, we analyzed both the most highly expressed genes and those that were most significantly different from the proliferative zone (PZ) as a single comparator. Confirmation of the differential expression of selected genes was done by quantitative real-time RT-PCR. A total of 8 transcripts showing high expression unique to the PC included translationally-controlled tumor protein (Tpt1), connective tissue growth factor (Ctgf), mortality factor 4 (Morf4l1), growth arrest specific 6 (Gas6), type V procollagen (Col5a2), frizzled-related protein (Frzb), GDP-dissociation inhibitor 2 (Gdi2) and Jun D proto-oncogene (Jund). In contrast, 8 transcripts showing unique high expression in the RZ included hyaluronan and proteoglycan link protein 1 (Hapln1), hemoglobin beta-2 subunit, type I procollagen (Col1a2), retinoblastoma binding protein 4 (LOC685491), Sparc-related modular calcium binding 2 (Smoc2), and calpastatin (Cast). Other genes were highly expressed in cells from both PC and RZ zones, including collagen II, collagen IX, catenin (cadherin associated protein) beta 1, eukaryotic translation elongation factor, high mobility group, ribosomal protein, microtubule-associated protein, reticulocalbin, thrombospondin, retinoblastoma binding protein, carboxypeptidase E, carnitine palmitoyltransferase 1, cysteine rich glycoprotein, plexin B2 (Plxnb2), and gap junction membrane channel protein. Functional classification of the most highly expressed transcripts were analyzed, and the pathway analysis indicated that ossification, bone remodeling, and cartilage development were uniquely enriched in the PC whereas both the PC and RZ showed pathway enrichment for skeletal development, extracellular matrix structural constituent, proteinaceous extracellular matrix, collagen, extracellular matrix, and extracellular matrix part pathways. We conclude that differential gene expression exists between the RZ and PC chondrocytes and these differentially expressed genes have unique roles to play corresponding to the function of their respective zones.

Temporal Changes in PTHrP, Bcl-2, Bax, Caspase, TGF-β, and FGF-2 Expression Following Growth Plate Irradiation With or Without Radioprotectant

This study examined temporal changes in growth plate apoptosis molecules and growth factors in an animal model of radiation injury with and without a radioprotectant. Thirty weanling 5-week Sprague-Dawley rats underwent right knee irradiation with single-fraction 17.5 Gy while the left served as internal control. Six animals each were sacrificed at 0.5, 1, 2, 3, or 4 weeks after irradiation. Half of the animals received pretreatment with amifostine (WR-2721) radioprotectant. Immunohistochemical staining for PTHrP, Bcl-2, Bax, caspase-3, FGF-2, and TGF-β was performed. PTHrP decreased to a nadir at 1 week after irradiation but rebounded to above control levels at 2 weeks in the reserve and transitional zones. The radioprotectant amifostine blunted the decrease in PTHrP but kept PTHrP expression lower than controls during the rebound phase in untreated irradiated animals. Hypertrophic zone Bax expression was decreased by amifostine in both irradiated and non-irradiated limbs at 1 and 2 weeks. FGF, TGF-β, Bcl-2, and caspase levels generally decreased at 1 week and returned thereafter toward control levels. These findings underscore the importance of PTHrP in response to growth plate irradiation and show the novel finding of a decrease in Bax expression with amifostine pretreatment.

Novel radioprotectant drugs for sparing radiation-induced damage to the physis.

Purpose: To determine if pentoxifylline, interleukin 1α, selenium and misoprostol can minimize damage to physeal longitudinal growth during single radiation dose exposure in an animal model. Materials and methods: Eighty‐seven weanling Sprague–Dawley rats were randomized into 15 drug/dose groups. All groups received a single 17.5‐Gy gamma‐irradiation exposure to the right knee, the left limb serving as an internal control. Pentoxifylline was injected 30 min before exposure, sodium selenite and interleukin 1α 24 h before exposure and misoprostol 2 h before exposure. Positive controls received 17.5 Gy. At 6 weeks, animals were sacrificed, the hind limb lengths were measured and detailed histomorphometric analysis was performed. Results: Statistically significant reductions (p≤0.03) in mean limb length discrepancy compared with irradiation alone were seen following administration of pentoxifylline (50 mg kg−1), interleukin 1α (15 mcg kg−1), selenium (5 mg kg−1) and misoprostol (20 mg kg−1). Histomorphometric endpoints and growth rate remained altered at 6 weeks despite treatment, but length discrepancy reduction was highly correlated with the appearance of regenerative clones. Conclusions: Each drug reduced the amount of anticipated growth arrest in the animal model and some compared favourably in magnitude with that previously demonstrated for the established radioprotectant drug amifostine. Restoration of growth appears related to appearance of regenerative clones.

Heparin Modulates Integrin-Mediated Cellular Adhesion: Specificity of Interactions with α and β Integrin Subunits

Heparin is known to influence the growth, proliferation, and migration of vascular cells, but the precise mechanisms are unknown. We previously demonstrated that unfractionated heparin (UH) binds to the platelet integrin αIIbβ3, and enhances ligand binding. To help define the specificity and site(s) of heparin-integrin interactions, we employed the erythroleukemic K562 cell line, transfected to express specific integrins (αvβ3, αvβ5, and αIIbβ3). By comparing K562 cells expressing a common α subunit (Kαvβ3, Kαvβ5) with cells expressing a common β subunit (Kαvβ3, KαIIbβ3), we observed that heparin differentially modulated integrin-mediated adhesion to vitronectin. UH at 0.5–7.5 μg/ml consistently enhanced the adhesion of β3expressing cells (Kαvβ3,KαIIbβ3). In contrast, UH at 0.5–7.5 μg/ml inhibited Kαvβ5adhesion. Experiments using integrin-blocking antibodies, appropriate control ligands, and nontransfected native K562 cells revealed that heparins actions were mediated by the specific integrins under study. Preincubation of heparin with Kαvβ3cells enhanced adhesion, while preincubation of heparin with the adhesive substrate (vitronectin) had minimal effect. There was a structural specificity to heparins effect, in that a low molecular weight heparin and chondroitin sulfate showed significantly less enhancement of adhesion. These findings suggest that heparins modulation of integrin-ligand interactions occurs through its action on the integrin. The inhibitory or stimulatory effects of heparin depend on the β subunit type, and the potency is dictated by structural characteristics of the glycosaminoglycan.

Precision and Accuracy of DXA and pQCT for Densitometry of the Rat Femur

Measurements of bone mineral density and bone mineral content are key data in the study of osteoporosis and pathologic skeletal disease. Dual-energy X-ray absorptiometry and peripheral quantitative computed tomography are used in human and small animal studies. The purpose of this study was to evaluate the precision, accuracy, and systematic bias of measurement of the rat femur. Comparing machine-measured parameters with standard, nonradiographic measurements, we assessed validation of relative and absolute accuracy. Regression analysis and calculations of percent difference from standard values were used to determine the accuracy of each densitometry technique. Machine-specific and subject-specific precision was evaluated for each densitometer using repeated scans to calculate coefficients of variation. Each of the methods of densitometry examined in this study produced comparable results and was sensitive to small changes following experimental stimuli. Further, our assessment of the precision and accuracy observed between methods of scanning excised rat femurs validates our data acquisition method and serves as a foundation for future densitometry studies.

Effects of Radiation Therapy on Chondrocytes In Vitro

The negative irradiation complications of growth loss leading to limb length asymmetry and pathological fracture incurred following radiation therapy in pediatric patients has led to a renewed interest in understanding the specific effects of irradiation on the growth plate and the surrounding bone. In the present report, we examined the radiation therapy effects on primary rat growth cartilage chondrocytes in order to determine the chondrocyte radiosensitivity relative to other bone cell constituents and tumor cells, the postirradiation temporal progression of radiation-induced alterations in chondrocyte function, and the time course for the functional restoration of chondrocyte pathways that drive the eventual recovery in growth function. We employed an in vitro primary rat costochondral growth cartilage cell culture model system to evaluate the radiation therapy effects on proliferative chondrocytes using serial radiation doses (0–20 Gy) that are well within the clinically relevant range. Following irradiation, all of the following occurred in a dose-dependent manner: proliferation decreased, cytotoxicity increased, several markers of apoptosis increased, markers of radiation-induced cellular differentiation increased, and cell synthetic activity was disturbed. Alterations in proliferation, cell death, and induction of apoptosis are likely due to a transient radiation-induced derangement of the parathyroid hormone-related protein-Indian hedgehog proliferation-maturation pathway. Alterations in cellular differentiation and cell synthetic activity are novel observations for chondrocytes. Further, these results correspond very well to our previous work in an in vivo Sprague-Dawley rat model, making this model particularly relevant to researching the radiation therapy effects on longitudinal growth.

Transforming Growth Factor Alpha is a Critical Mediator of Radiation Lung Injury

Radiation fibrosis of the lung is a late toxicity of thoracic irradiation. Epidermal growth factor (EGF) signaling has previously been implicated in radiation lung injury. We hypothesized that TGF-α, an EGF receptor ligand, plays a key role in radiation-induced fibrosis in lung. Mice deficient in transforming growth factor (TGF-α–/–) and control C57Bl/6J (C57-WT) mice were exposed to thoracic irradiation in 5 daily fractions of 6 Gy. Cohorts of mice were followed for survival (n ≥ 5 per group) and tissue collection (n = 3 per strain and time point). Collagen accumulation in irradiated lungs was assessed by Massons trichrome staining and analysis of hydroxyproline content. Cytokine levels in lung tissue were assessed with ELISA. The effects of TGF-α on pneumocyte and fibroblast proliferation and collagen production were analyzed in vitro. Lysyl oxidase (LOX) expression and activity were measured in vitro and in vivo. Irradiated C57-WT mice had a median survival of 24.4 weeks compared to 48.2 weeks for irradiated TGF-α–/– mice (P = 0.001). At 20 weeks after irradiation, hydroxyproline content was markedly increased in C57-WT mice exposed to radiation compared to TGF-α–/– mice exposed to radiation or unirradiated C57-WT mice (63.0, 30.5 and 37.6 μg/lung, respectively, P = 0.01). C57-WT mice exposed to radiation had dense foci of subpleural fibrosis at 20 weeks after exposure, whereas the lungs of irradiated TGF-α –/– mice were largely devoid of fibrotic foci. Lung tissue concentrations of IL-1β, IL-4, TNF-α, TGF-β and EGF at multiple time points after irradiation were similar in C57-WT and TGF-α–/– mice. TGF-α in lung tissue of C57-WT mice rose rapidly after irradiation and remained elevated through 20 weeks. TGF-α–/– mice had lower basal LOX expression than C57-WT mice. Both LOX expression and LOX activity were increased after irradiation in all mice but to a lesser degree in TGF-α–/– mice. Treatment of NIH-3T3 fibroblasts with TGF-α resulted in increases in proliferation, collagen production and LOX activity. These studies identify TGF-α as a critical mediator of radiation-induced lung injury and a novel therapeutic target in this setting. Further, these data implicate TGF-α as a mediator of collagen maturation through a TGF-β independent activation of lysyl oxidase.

Radioprotectant combinations spare radiation-induced damage to the physis more than fractionation alone.

Purpose: The aim of this study was to determine if fractionation and individual or combinations of radioprotectants could minimize damage to physeal longitudinal growth in an animal model to any greater extent than fractionation alone. Materials and methods: Sixty-three weanling male Sprague-Dawley rats were randomized into seven equal groups. Five groups received a total 25 Gy radiation exposure in three equal fractions to the right knee with the left as non-irradiated control. For each group, pentoxifylline, misoprostol, and amifostine were given individually and amifostine was also given in combination with each of the other drugs prior to the radiation fractions. One group each received 25 Gy in one or three fractions without radioprotection. At six weeks, limb lengths and histomorphometry were assessed. Results: The single fraction of 25 Gy caused a mean tibial length discrepancy of 24.4%. Fractionation decreased this to 18.8% (p < 0.001). Beyond fractionation alone, the mean femoral length discrepancies were significantly decreased by each of the added individual and combination radioprotectant drugs (p < 0.0004). The smallest absolute femoral length discrepancy (11%) was achieved with fractionation and the combination of amifostine and misoprostol. Conclusions: Radioprotectants may be beneficial in growth plate radioprotection, alone or in combination.