Integration of molecular data with histologic, radiologic, and clinical features is imperative for accurate diagnosis of pediatric central nervous system (CNS) tumors. Whole transcriptome RNA ...sequencing (RNAseq), a genome-wide and non-targeted approach, allows for the detection of novel or rare oncogenic fusion events that contribute to the tumorigenesis of a substantial portion of pediatric low- and high-grade glial and glioneuronal tumors. We present two cases of pediatric glioneuronal tumors occurring in the occipital region with a CLIP2::MET fusion detected by RNAseq. Chromosomal microarray studies revealed copy number alterations involving chromosomes 1, 7, and 22 in both tumors, with Case 2 having an interstitial deletion breakpoint in the CLIP2 gene. By methylation profiling, neither tumor had a match result, but both clustered with the low-grade glial/glioneuronal tumors in the UMAP. Histologically, in both instances, our cases displayed characteristics of a low-grade tumor, notably the absence of mitotic activity, low Ki-67 labeling index and the lack of necrosis and microvascular proliferation. Glial and neuronal markers were positive for both tumors. Clinically, both patients achieved clinical stability post-tumor resection and remain under regular surveillance imaging without adjuvant therapy at the last follow-up, 6 months and 3 years, respectively. This is the first case report demonstrating the presence of a CLIP2::MET fusion in two pediatric low-grade glioneuronal tumors (GNT). Conservative clinical management may be considered for patients with GNT and CLIP2:MET fusion in the context of histologically low-grade features.
The endothelium is a metabolically active secretory tissue, capable of responding to a wide array of environmental stimuli. Hypoxia and vascular endothelial growth factor (VEGF) are two components of ...the putative fracture microenvironment. This study investigated the role of hypoxia and VEGF on endothelial cell activation as it relates to the bone repair process. It was hypothesized that endothelial cells may have an important osteogenic role in fracture healing through the production of bone morphogenetic protein-2 (BMP-2), an osteogenic cytokine at the fracture site. Therefore, BMP-2 mRNA and protein expression in endothelial cells under hypoxia and/or VEGF treatment was studied. The authors observed a 2-fold to 3-fold up-regulation of BMP-2 mRNA expression in bovine capillary endothelial cells and human microvascular endothelial cells stimulated with hypoxia or rhVEGF. Furthermore, the combined effects of hypoxia and rhVEGF appeared to be additive on BMP-2 mRNA expression in bovine capillary endothelial cells. Actinomycin D and cycloheximide studies suggested that the increased mRNA expression was transcriptionally regulated. BMP-2 protein expression was up-regulated after 24 and 48 hours of treatment with either hypoxia or rhVEGF in bovine capillary endothelial cells. Surprisingly, the data suggest that endothelial cells may play not only an angiogenic role but also an osteogenic role by a direct stimulation of the osteoblasts, through the enhanced expression of a potent osteogenic factor, BMP-2, at the fracture site. (Plast. Reconstr. Surg. 1092384, 2002.)
The well-described detrimental effects of ionizing radiation on the regeneration of bone within a fracture site include decreased osteocyte number, suppressed osteoblast activity, and diminished ...vascularity. However, the biologic mechanisms underlying osteoradionecrosis and the impaired fracture healing of irradiated bone remain undefined. Ionizing radiation may decrease successful osseous repair by altering cytokine expression profiles resulting from or leading to a change in the osteoblastic differentiation state. These changes may, in turn, cause alterations in osteoblast proliferation and extracellular matrix formation. The purpose of this study was to investigate the effects of ionizing radiation on the proliferation, maturation, and cytokine production of MC3T3-E1 osteoblast-like cells in vitro. Specifically, the authors examined the effects of varying doses of ionizing radiation (0, 40, 400, and 800 cGy) on the expression of transforming growth factor-beta1 (TGF-beta1), vascular endothelial growth factor (VEGF), and alkaline phosphatase. In addition, the authors studied the effects of ionizing radiation on MC3T3-E1 cellular proliferation and the ability of conditioned media obtained from control and irradiated cells to regulate the proliferation of bovine aortic endothelial cells. Finally, the authors evaluated the effects of adenovirus-mediated TGF-beta1 gene therapy in an effort to "rescue" irradiated osteoblasts. The exposure of osteoblast-like cells to ionizing radiation resulted in dose-dependent decreases in cellular proliferation and promoted cellular differentiation (i.e., increased alkaline phosphatase production). Additionally, ionizing radiation caused dose-dependent decreases in total TGF-beta1 and VEGF protein production. Decreases in total TGF-beta1 production were due to a decrease in TGF-beta1 production per cell. In contrast, decreased total VEGF production was secondary to decreases in cellular proliferation, because the cellular production of VEGF by irradiated osteoblasts was moderately increased when VEGF production was corrected for cell number. Additionally, in contrast to control cells (i.e., nonirradiated), conditioned media obtained from irradiated osteoblasts failed to stimulate the proliferation of bovine aortic endothelial cells. Finally, transfection of control and irradiated cells with a replication-deficient TGF-beta1 adenovirus before irradiation resulted in an increase in cellular production of TGF-beta1 protein and VEGF. Interestingly, this intervention did not alter the effects of irradiation on cellular proliferation, which implies that alterations in TGF-beta1 expression do not underlie the deficiencies noted in cellular proliferation. The authors hypothesize that ionizing radiation-induced alterations in the cytokine profiles and differentiation states of osteoblasts may provide insights into the cellular mechanisms underlying osteoradionecrosis and impaired fracture healing.
Laboratory of Developmental Biology and Repair and Department of
Surgery, New York University Medical Center, New York, New York,
10016
Angiogenesis is essential for the
increased delivery of oxygen ...and nutrients required for the reparative
processes of bone healing. Vascular endothelial growth factor (VEGF), a
potent angiogenic growth factor, has been implicated in this process.
We have previously shown that hypoxia specifically and potently
regulates the expression of VEGF by osteoblasts. However, the molecular
mechanisms governing this interaction remain unknown. In this study, we
hypothesized that the hypoxic regulation of VEGF expression by
osteoblasts occurs via an oxygen-sensing mechanism similar to the
regulation of the erythropoietin gene (EPO). To test this hypothesis,
we examined the kinetics of oxygen concentration on osteoblast VEGF expression. In addition, we analyzed the effects of nickel and cobalt
on the expression of VEGF in osteoblastic cells because these metallic
ions mimic hypoxia by binding to the heme portion of oxygen-sensing
molecules. Our results indicated that hypoxia potently stimulates VEGF
mRNA expression. In addition, we found that nickel and cobalt both
stimulate VEGF gene expression in a similar time- and dose-dependent
manner, suggesting the presence of a hemelike oxygen-sensing mechanism
similar to that of the EPO gene. Moreover, actinomycin D,
cycloheximide, dexamethasone, and mRNA stabilization studies
collectively established that this regulation is predominantly
transcriptional, does not require de novo protein synthesis, and is not
likely mediated by the transcriptional activator AP-1. These studies
demonstrate that hypoxia, nickel, and cobalt regulate VEGF expression
in osteoblasts via a similar mechanism, implicating the involvement of
a heme-containing oxygen-sensing molecule. This may represent an
important mechanism of VEGF regulation leading to increased
angiogenesis in the hypoxic microenvironment of healing bone.
bone repair; osteogenesis; angiogenesis
Gain-of-function mutations in fibroblast growth factor receptors have been identified in numerous syndromes associated with premature cranial suture fusion. Murine models in which the posterior ...frontal suture undergoes programmed fusion after birth while all other sutures remain patent provide an ideal model to study the biomolecular mechanisms that govern cranial suture fusion. Using adenoviral vectors and targeted
in utero injections in rats, we demonstrate that physiological posterior frontal suture fusion is inhibited using a dominant-negative fibroblast growth factor receptor-1 construct, whereas the normally patent coronal suture fuses when infected with a construct that increases basic fibroblast growth factor biological activity. Our data may facilitate the development of novel, less invasive treatment options for children with craniosynostosis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Angiogenesis is essential to both normal and pathological bone physiology. Vascular endothelial growth factor (VEGF) has been implicated in angiogenesis, whereas transforming growth factor-β1 ...(TGF-β1) modulates bone differentiation, matrix formation, and cytokine expression. The purpose of this study was to investigate the relationship between TGF-β1 and VEGF expression in osteoblasts and osteoblast-like cells. Northern blot analysis revealed an early peak of VEGF mRNA (6-fold at 3 h) in fetal rat calvarial cells and MC3T3-E1 osteoblast-like cells after stimulation with TGF-β1 (2.5 ng/ml). The stability of VEGF mRNA in MC3T3-E1 cells was not increased after TGF-β1 treatment. Actinomycin D inhibited the TGF-β1-induced peak in VEGF mRNA, whereas cycloheximide did not. Blockade of TGF-β1 signal transduction via a dominant-negative receptor II adenovirus significantly decreased TGF-β1 induction of VEGF mRNA. Additionally, TGF-β1 induced a dose-dependent increase in VEGF protein expression by MC3T3-E1 cells ( P < 0.01). Dexamethasone similarly inhibited VEGF protein expression. Both TGF-β1 mRNA and VEGF mRNA were concurrently present in rat membranous bone, and both followed similar patterns of expression during rat mandibular fracture healing (mRNA and protein). In summary, TGF-β1-induced VEGF expression by osteoblasts and osteoblast-like cells is a dose-dependent event that may be intimately related to bone development and fracture healing.
Despite its prevalence, the etiopathogenesis of craniosynostosis is poorly understood. To better understand the biomolecular events that occur when normal craniofacial growth development goes awry, ...we must first investigate the mechanisms of normal suture fusion. Murine models in which the posterior frontal (PF) suture undergoes programmed sutural fusion shortly after birth provide an ideal model to study these mechanisms. In previous studies, our group and others have shown that sutural fate (i.e., fusion vs. patency) is regulated by the dura mater (DM) directly underlying a cranial suture. These studies have led to the hypothesis that calvarial DM is regionally differentiated and that this differentiation guides the development of the overlying suture. To test this hypothesis, we evaluated the messenger RNA (mRNA) expression of osteogenic cytokines (transforming growth factor β1 TGF‐β1 and TGF‐β3) and bone‐associated extracellular matrix (ECM) molecules (collagen I, collagen III, osteocalcin, and alkaline phosphatase) in freshly isolated, rat dural tissues associated with the PF (programmed to fuse) or sagittal (SAG; remains patent) sutures before histological evidence of sutural fusion (postnatal day 6 N6). In addition, osteocalcin protein expression and cellular proliferation were localized using immunohistochemical staining and 5‐bromo‐2′deoxyuridine (BrdU) incorporation, respectively. We showed that the expression of osteogenic cytokines and bone‐associated ECM molecules is potently up‐regulated in the DM associated with the PF suture. In addition, we showed that cellular proliferation in the DM associated with the fusing PF suture is significantly less than that found in the patent SAG suture just before the initiation of sutural fusion N6. Interestingly, no differences in cellular proliferation rates were noted in younger animals (embryonic day 18 E18 and N2). To further analyze regional differentiation of cranial suture‐associated dural cells, we established dural cell cultures from fusing and patent rat cranial sutures in N6 rats and evaluated the expression of osteogenic cytokines (TGF‐β1 and fibroblast growth factor 2 FGF‐2) and collagen I. In addition, we analyzed cellular production of proliferating cell nuclear antigen (PCNA). These studies confirmed our in vivo findings and showed that dural cell cultures derived from the fusing PF suture expressed significantly greater amounts of TGF‐β1, FGF‐2, and collagen I. In addition, similar to our in vivo findings, we showed that PF suture‐derived dural cells produced significantly less PCNA than SAG suture‐derived dural cells. Finally, coculture of dural cells with fetal rat calvarial osteoblastic cells (FRCs) revealed a statistically significant increase in proliferation (p < 0.001) in FRCs cocultured with SAG suture‐derived dural cells as compared with FRCs cocultured alone or with PF suture‐derived dural cells. Taken together, these data strongly support the hypothesis that the calvarial DM is regionally differentiated resulting in the up‐regulation of osteogenic cytokines and bone ECM molecules in the dural tissues underlying fusing but not patent cranial sutures. Alterations in cytokine expression may govern osteoblastic differentiation and ECM molecule deposition, thus regulating sutural fate. Elucidation of the biomolecular events that occur before normal cranial suture fusion in the rat may increase our understanding of the events that lead to premature cranial suture fusion.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
1 Laboratory of Developmental Biology and Repair, Department
of Surgery, New York University School of Medicine, New York, New
York 10016; and 2 Department of Surgery, University of
Connecticut, ...Farmington, Connecticut 06032
Angiogenesis,
the formation of new blood vessels, is crucial to the process of
fracture healing. Vascular disruption after osseous injury results in
an acidic, hypoxic wound environment. We have previously shown that
osteoblasts can produce vascular endothelial growth factor (VEGF) in
response to a variety of stimuli. In this study we examined pH and
lactate concentration, two components of the putative fracture
extracellular microenvironment, and determined their relative
contribution to regulation of rat calvarial osteoblast VEGF production
under both normoxic and hypoxic conditions. Our results demonstrate
that pH and lactate concentration do independently affect osteoblast
VEGF mRNA and protein production. Acidic pH (7.0) significantly
decreased VEGF production, under normoxic and hypoxic conditions
( P < 0.05), compared with neutral pH (7.4). This
decrease was primarily transcriptionally regulated, because the rate of
VEGF mRNA degradation was unchanged at pH 7.0 vs. 7.4. Similarly, an
elevated lactate concentration (22 mM) also depressed osteoblast
elaboration of VEGF at both neutral and acidic pH ( P < 0.001). Furthermore, the effects of increasing acidity and elevated
lactate appeared to be additive.
pH; lactate; hypoxia; angiogenesis; bone
The dura mater, the outermost layer of the meninges, is thought to be essential for calvarial morphogenesis, postnatal suture fusion, and osseous repair of calvarial defects. Despite numerous studies ...illustrating the fundamental role of the dura mater, there is little information about the autocrine and paracrine mechanisms regulating dural cell biology during calvarial ossification. Previous work conducted in the authorsʼ laboratory demonstrated that non-suture-associated dural cells from 6-day-old rat pups expressed high levels of fibroblast growth factor 2 (FGF-2), whereas dural cells from 60-day-old adult rats expressed very little FGF-2. Because young mammals can successfully heal large calvarial defects, the authors sought to investigate the autocrine and/or paracrine effects of FGF-2 on the proliferation, gene expression, and alkaline phosphatase production of dural cells.Cultures of non-suture-associated dural cells were established from 6-day-old Sprague-Dawley rat pups and then stimulated with recombinant human FGF-2 (rhFGF-2; 10 ng/ml). Dural cells stimulated with rhFGF-2 proliferated significantly faster than untreated dural cells at 24 hours (2.1 × 10 ± 3.2 × 10 versus 1.1 × 10 ± 1.8 × 10, p ≤ 0.001) and 48 hours (2.3 × 10 ± 4.2 × 10 versus 1.2 × 10 ± 1.3 × 10, p ≤ 0.001). Moreover, dural cells stimulated with rhFGF-2 expressed 7-fold more proliferating cell nuclear antigen than did control cultures. Treatment with rhFGF-2 increased dural cell expression of genes important for skeletal repairFGF-2 (7-fold), transforming growth factor β1 (3-fold), transforming growth factor β3 (4-fold), and type I collagen (4-fold). Furthermore, rhFGF-2 increased dural cell expression of osteopontin (2-fold), a “late” marker of osteoblastic differentiation. Interestingly, dural cell alkaline phosphatase activity, an “earlier” marker of osteoblast differentiation, was significantly decreased by treatment with rhFGF-2 compared with control cultures at 24 hours (0.005 ± 0.001 versus 0.01 ± 0.003, p ≤ 0.01) and 48 hours (0.004 ± 0.0009 versus 0.01 ± 0.0009). Together these data provide insight into the autocrine and paracrine effects of FGF-2 on the biology of the dura mater. (Plast. Reconstr. Surg. 109645, 2002.)
tk;2For the reconstructive plastic surgeon, knowledge of the molecular biology underlying membranous fracture healing is becoming increasingly vital. Understanding the complex patterns of gene ...expression manifested during the course of membranous fracture repair will be crucial to designing therapies that augment poor fracture healing or that expedite normal osseous repair by strategic manipulation of the normal course of gene expression. In the current study, we present a rat model of membranous bone repair. This model has great utility because of its technical simplicity, reproducibility, and relatively low cost. Furthermore, it is a powerful tool for analysis of the molecular regulation of membranous bone repair by immunolocalization and/or in situ hybridization techniques.In this study, an osteotomy was made within the caudal half of the hemimandible, thus producing a stable bone defect without the need for external or internal fixation. The healing process was then catalogued histologically in 28 Sprague-Dawley rats that were serially killed at 1, 2, 3, 4, 5, 6, and 8 weeks after operation. Furthermore, using this novel model, we analyzed, within the context of membranous bone healing, the temporal and spatial expression patterns of several members of the bone morphogenetic protein (BMP) family, known to be critical regulators of cells of osteoblast lineage. Our data suggest that BMP-2/-4 and BMP-7, also known as osteogenic protein-1 (OP-1), are expressed by osteoblasts, osteoclasts, and other more primitive mesenchymal cells within the fracture callus during the early stages of membranous fracture healing. These proteins continue to be expressed during the process of bone remodeling, albeit less prominently. The return of BMP-2/-4 and OP-1 immunostaining to baseline intensity coincides with the histological appearance of mature lamellar bone. Taken together, these data underscore the potentially important regulatory role played by the bone morphogenetic proteins in the process of membranous bone repair. (Plast. Reconstr. Surg. 107124, 2001.)
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