Hongwei Cheng

Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs).

Background:Bone morphogenic proteins (BMPs) are known to promote osteogenesis, and clinical trials are currently underway to evaluate the ability of certain BMPs to promote fracture-healing and spinal fusion. The optimal BMPs to be used in different clinical applications have not been elucidated, an

Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery.

Efficacious bone regeneration could revolutionize the clinical management of bone and musculoskeletal disorders. Although several bone morphogenetic proteins (BMPs) (mostly BMP-2 and BMP-7) have been shown to induce bone formation, it is unclear whether the currently used BMPs represent the most osteogenic ones. Until recently, comprehensive analysis of osteogenic activity of all BMPs has been hampered by the fact that recombinant proteins are either not biologically active or not available for all BMPs. In this study, we used recombinant adenoviruses expressing the 14 types of BMPs (AdBMPs), and demonstrated that, in addition to currently used BMP-2 and BMP-7, BMP-6 and BMP-9 effectively induced orthotopic ossification when either AdBMP-transduced osteoblast progenitors or the viral vectors were injected into the quadriceps of athymic mice. Radiographic and histological evaluation demonstrated that BMP-6 and BMP-9 induced the most robust and mature ossification at multiple time points. BMP-3, a negative regulator of bone formation, was shown to effectively inhibit orthotopic ossification induced by BMP-2, BMP-6, and BMP-7. However, BMP-3 exerted no inhibitory effect on BMP-9-induced bone formation, suggesting that BMP-9 may transduce osteogenic signaling differently. Our findings suggest that BMP-6 and BMP-9 may represent more effective osteogenic factors for bone regeneration.

Potential use of Sox9 gene therapy for intervertebral degenerative disc disease.

Study Design. A new recombinant adenoviral vector expressing Sox9, a chondrocyte-specific transcription factor, was tested in a chondroblastic cell line and primary human intervertebral disc cells in vitro. Direct infection of intervertebral disc cells then was assessed in a rabbit model. Objectives. To deliver a potentially therapeutic viral vector expressing Sox9 to degenerative human and rabbit intervertebral discs cells, and to assess the effect of Sox9 expression on Type 2 collagen production. Summary of the Background Data. The concentration of competent Type 2 collagen, an essential constituent of the healthy nucleus pulposus, declines with intervertebral disc degeneration. Recent studies suggest that Sox9 upregulates Type 2 collagen production. Interventions that augment Type 2 collagen production by intervertebral disc cells may represent a novel therapeutic method for patients with degenerative disc disease. Methods. Adenoviral delivery vectors expressing Sox9 and green fluorescent protein were constructed using the AdEasy system. The chondroblastic cell line, HTB-94, and cultured human degenerated intervertebral disc cells were infected with the vectors. Reverse transcriptase-polymerase chain reaction and immunohistochemical analyses were performed to document increased Type 2 collagen expression. The AdSox9 virus then was injected directly into the intervertebral discs of three rabbits. After 5 weeks, the injected discs were evaluated histologically. Results. The AdSox9 virus efficiently transduced HTB-94 cells and degenerated human disc cells. Western blot analysis confirmed increased Sox9 production. Increased Type 2 collagen production was demonstrated in infected HTB-94 and human disc cells using both reverse transcriptase-polymerase chain reaction and immunohistochemical staining. In the rabbit model, cells infected with AdSox9 maintained a chondrocytic phenotype, and the architecture of the nucleus pulposus was preserved over a 5-week study period compared to control discs. Conclusions. A novel adenoviral vector efficiently increased Sox9 and Type 2 collagen synthesis in cultured chondroblastic cells and human degenerated disc cells. In a rabbit model, sustained Sox9 production preserved the histologic appearance of the nucleus pulposus cells in vivo. These findings suggest a potential role for Sox9 gene therapy in the treatment of human degenerative disc disease.

Transcriptional Characterization of Bone Morphogenetic Proteins (BMPs)-Mediated Osteogenic Signaling

Bone formation is presumably a complex and well‐orchestrated process of osteoblast lineage‐specific differentiation. As members of the TGFβ superfamily, bone morphogenetic proteins (BMPs) play an important role in regulating osteoblast differentiation and subsequent bone formation. Several BMPs are able to induce de novo bone formation. Although significant progress has recently been made about the transcriptional control of osteoblast differentiation, detailed molecular events underlying the osteogenic process remain to be elucidated. In order to identify potentially important signaling mediators activated by osteogenic BMPs but not by non‐osteogenic BMPs, we sought to determine the transcriptional differences between three osteogenic BMPs (i.e., BMP‐2, BMP‐6, and BMP‐9) and two inhibitory/non‐osteogenic BMPs (i.e., BMP‐3 and BMP‐12). Through the microarray analysis of approximately 12,000 genes in pre‐osteoblast progenitor cells, we found that expression level of 203 genes (105 up‐regulated and 98 down‐regulated) was altered >2‐fold upon osteogenic BMP stimulation. Gene ontology analysis revealed that osteogenic BMPs, but not inhibitory/non‐osteogenic BMPs, activate genes involved in the proliferation of pre‐osteoblast progenitor cells towards osteoblastic differentiation, and simultaneously inhibit myoblast‐specific gene expression. BMP‐regulated expression of the selected target genes was confirmed by RT‐PCR, as well as by the CodeLink Bioarray analysis. Our findings are consistent with the notion that osteogenesis and myogenesis are two divergent processes. Further functional characterization of these downstream target genes should provide important insights into the molecular mechanisms behind BMP‐mediated bone formation.

Cytoplasmic and/or nuclear accumulation of the β‐catenin protein is a frequent event in human osteosarcoma

The molecular events that precede the development of osteosarcoma, the most common primary malignancy of bone, are unclear, and concurrent molecular and genetic alterations associated with its pathogenesis have yet to be identified. Recent studies suggest that activation of β‐catenin signaling may play an important role in human tumorigenesis. To investigate the potential role of β‐catenin deregulation in human osteosarcoma, we analyzed a panel of 47 osteosarcoma samples for β‐catenin accumulation using immunohistochemistry. Potential activating mutations were investigated by sequencing exon 3 of the β‐catenin gene in genomic DNA isolated from tumor samples. Our findings revealed cytoplasmic and/or nuclear accumulation of β‐catenin in 33 of 47 samples (70.2%); however, mutation analysis failed to detect any genetic alterations within exon 3, suggesting that other regulatory mechanisms may play an important role in activating β‐catenin signaling in osteosarcoma. In our survival analysis, β‐catenin deregulation conferred a hazard ratio of 1.05, indicating that β‐catenin accumulation does not appear to be of prognostic value for osteosarcoma patients. When analyzed against other clinicopathologic parameters, β‐catenin accumulation correlated only with younger age at presentation (26.4 vs. 39.8 years). Nevertheless, our results demonstrate that the deregulation of β‐catenin signaling is a common occurrence in osteosarcoma that is implicated in the pathogenesis of osteosarcoma.

Adenoviral Vector-Mediated Gene Transfer for Human Gene Therapy

Human gene therapy promises to change the practice of medicine by treating the causes of disease rather than the symptoms. Since the first clinical trial made its debut ten years ago, there are over 400 approved protocols in the United States alone, most of which have failed to show convincing data of clinical efficacy. This setback is largely due to the lack of efficient and adequate gene transfer vehicles. With the recent progress in elucidating the molecular mechanisms of human diseases and the imminent arrival of the post genomic era, there are increasing numbers of therapeutic genes or targets that are available for gene therapy. Therefore, the urgency and need for efficacious gene therapies are greater than ever. Clearly, the current fundamental obstacle is to develop delivery vectors that exhibit high efficacy and specificity of gene transfer. Recombinant adenoviruses have provided a versatile system for gene expression studies and therapeutic applications. Of late, there has been a remarkable increase in adenoviral vector-based clinical trials. Recent endeavors in the development of recombinant adenoviral vectors have focused on modification of virus tropism, accommodation of larger genes, increase in stability and control of transgene expression, and down-modulation of host immune responses. These modifications and continued improvements in adenoviral vectors will provide a great opportunity for human gene therapy to live up to its enormous potential in the second decade.

Tyrosine kinase inhibitor STI-571/gleevec down-regulates the β-catenin signaling activity

Beta-Catenin is a critical transducer of the Wnt signal pathway and plays an important role in many developmental and cellular processes. Deregulation of beta-catenin signaling has been observed in a broad range of human tumors. In this report, we investigated whether tyrosine kinase inhibitor STI-571 could inhibit the beta-catenin signaling activity and hence suppress cell proliferation. Our results demonstrated that STI-571 effectively inhibited the constitutive activity of beta-catenin signaling in human colon cancer cells as well as the Wnt1-induced activation of beta-catenin signaling in HOS, HTB-94, and HEK 293 cells. Furthermore, STI-571 was shown to effectively suppress the proliferation of human colon cancer cells. Finally, we demonstrated that the Wnt1-mediated activation of a GAL4-beta-catenin heterologous transcription system was effectively inhibited by STI-571. Thus, our findings suggest that tyrosine phosphorylation may play an important role in regulating beta-catenin signaling activity, and inhibition of this signaling pathway by STI-571 may be further explored as an important target for alternative/adjuvant treatments for a broader range of human cancer.

8:42 An in vivo study of Sox9 gene therapy for intervertebral disc degeneration

Abstract Purpose of study: The concentration of type II collagen, an essential constituent of the healthy nucleus pulposus, declines with intervertebral disc degeneration. Interventions that stimulate type II collagen production by disc cells may represent a novel therapy for the treatment of degenerative disc disease. Recent studies have shown that Sox9 is an essential transcription factor for type II collagen synthesis, and it has been termed a “master regulator” of chondrocyte phenotype. The goals of this study were to assess the effects of delivering a gene expressing Sox9 to cultured rabbit chondrocytes and in vivo in rabbit intervertebral discs. Methods used: Adenoviral vectors expressing Sox9 (AdSox9) and green fluorescent protein were constructed using the AdEasy system. Isolated New Zealand rabbit intervertebral disc cells were infected with the vectors. Reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical analysis were performed to document increased type II collagen expression. After an anterior transabdominal approach, the AdSox9 virus was directly injected into the lumbar discs of two live NZ rabbits. After 5 weeks, rabbits were sacrificed, and discs were evaluated with magnetic resonance imaging (MRI) and histological examination. of findings: The AdSox9 virus efficiently transduced rabbit disc cells (greater than 60%). Western blot analysis confirmed increased Sox9 production. Using a luciferase reporter gene driven by type II collagen promoter, we demonstrated that Sox9 induced Pro-alphaI(II) mRNA expression through interaction with its promoter. Sox9-mediated type II collagen production was detected using RT-PCR and immunohistochemical staining. In the rabbit model at 5 weeks postinjection, control discs showed degenerative changes consistent with annulotomy-induced degeneration. In the nucleus, cells became spindle-shaped fibrocytes and the extracellular matrix assumed a fibrous appearance. However, the disc injected with AdSox9 retained the chondroid appearance of normal nucleus pulposus and the cells maintained a chondrocytic phenotype. MRI demonstrated lesser grades of disc degeneration in the Sox9-treated discs. Relationship between findings and existing knowledge: Successful adenovirus-mediated transduction of Sox9 transcription factor with stimulation of type II collagen was demonstrated in rabbit disc cells. In an annulotomy model of disc degeneration, in vivo transduction with exogenous Sox9 decreased disc degeneration. Overall significance of findings: These findings suggest a potential role for Sox9 gene therapy in the treatment of degenerative disc disease. Disclosures: No disclosures. Conflict of interest: No conflicts.

2:20 The osteogenic activity of 14 types of bone morphogenic proteins

Abstract Purpose of study: Although several bone morphogenic proteins (BMPs) have been shown to enhance bone healing and spinal fusion, the optimal BMPs or combination of BMPs to promote fusion are unknown. This study was designed to comprehensively elucidate the distinct and potentially synergistic osteogenic activity of 14 types of BMPs at various stages of osteogenesis. Methods used: Recombinant adenoviruses expressing 14 human BMPs (ie, BMP-2 to BMP-15) were constructed and used to infect both pluripotent mesenchymal progenitor C3H10T1/2 cells and committed osteoblastic C2C12 cells. The osteogenic activity was determined by measuring the induction of alkaline phosphatase, osteocalcin and matrix mineralization on BMP stimulation. of findings: BMP-2, -4, -6, -7 and -9 significantly induced alkaline phosphatase activity in C2C12 osteoblastic cells. BMP-5, -8, -10, -11, -12 and -13 exerted weak induction of alkaline phosphatase. However, only BMP-2, -6 and -9 significantly induced alkaline phosphatase activity in C3H10T1/2 pluripotent cells, although weak induction by BMP4 and 7 was detected. Histochemical staining assays demonstrated that BMP-6 and -9 induced the greatest increase in alkaline phosphatase staining, whereas BMP-2 and -4 (and BMP-7 to lesser extent) induced a modest increase in alkaline phosphatase activity in C2C12 cells. BMP-2, -6 and -9 significantly induced osteocalcin expression (ie, a late marker of osteogenesis) in C3H10T1/2 cells, and BMP-4 and -7 slightly increased osteocalcin expression. In C2C12 cells osteocalcin expression was strongly induced by BMP-2, -4, -6, -7 and -9, whereas BMP-5, -10 and -14 slightly induced expression. Mineralized nodules were readily detected in the C3H10T1/2 cells infected with BMP-2, -6 and -9 vectors (and, to lesser extent, with BMP-4 -7 and -10). Finally, we observed strong synergistic effects among the BMPs that had an ability to activate the osteogenic markers. Interestingly, a strong synergistic effect on osteogenesis was detected in cells stimulated by BMP-5 plus -10, BMP-5 plus -12, BMP-5 plus -13, BMP-7 plus -10 or BMP-7 plus -13. Relationship between findings and existing knowledge: We have conducted a comprehensive analysis of the osteogenic activity of 14 types of BMPs in osteoblastic progenitor cells and demonstrated that BMP-2, -6 and -9 are the most potent osteogenic factors, whereas BMP-4 and -7 exhibit reasonably strong osteoinductive activity. Further, certain combinations of BMPs exert a strong synergy on osteogenesis. Overall significance of findings: These findings have important implications for the development of effective new formulas for bone healing and spine fusion. Disclosures: No disclosures. Conflict of interest: Tong-Chuan He, grant research support.