Biomedical subjects

Edward M Schwarz

Publications and source records attributed to Edward M Schwarz.

At least 19 recordsLinked to original sources

PubMed2006

Alteration of femoral bone morphology and density in COX-2-/- mice.

A role of COX-2 in pathological bone destruction and fracture repair has been established; however, few studies have been conducted to examine the involvement of COX-2 in maintaining bone mineral density and bone micro-architecture. In this study, we examined bone morphology in multiple trabecular and cortical regions within the distal and diaphyseal femur of 4-month-old wild-type and COX-2-/- mice using micro-computed tomography. Our results demonstrated that while COX-2-/- female mice had normal bone geometry and trabecular microarchitecture at 4 months of age, the male knockout mice displayed reduced bone volume fraction within the distal femoral metaphysis. Furthermore, male COX-2-/- mice had a significant reduction in cortical bone mineral density within the central cortical diaphysis and distal epiphysis and metaphysis. Consistent with the observed reduction in cortical mineral density, biomechanical testing via 4-point-bending showed that male COX-2-/- mice had a significant increase in postyield deformation, indicating a ductile bone phenotype in male COX-2-/- mice. In conclusion, our study suggests that genetic ablation of COX-2 may have a sex-related effect on cortical bone homeostasis and COX-2 plays a role in maintaining normal bone micro-architecture and density in mice.

Animals↗

PubMed2006

Transforming growth factor-beta stimulates cyclin D1 expression through activation of beta-catenin signaling in chondrocytes.

Transforming growth factor-beta (TGF-beta) plays an essential role in chondrocyte maturation. It stimulates chondrocyte proliferation but inhibits chondrocyte differentiation. In this study, we found that TGF-beta rapidly induced beta-catenin protein levels and signaling in murine neonatal sternal primary chondrocytes. TGF-beta-increased beta-catenin induction was reproduced by overexpression of SMAD3 and was absent in Smad3(-/-) chondrocytes treated with TGF-beta. SMAD3 inhibited beta-transducin repeat-containing protein-mediated degradation of beta-catenin and immunoprecipitated with beta-catenin following TGF-beta treatment. Both SMAD3 and beta-catenin co-localized to the nucleus after TGF-beta treatment. Although both TGF-beta and beta-catenin stimulated cyclin D(1) expression in chondrocytes, the effect of TGF-beta was inhibited with beta-catenin gene deletion or SMAD3 loss of function. These results demonstrate that TGF-beta stimulates cyclin D(1) expression at least in part through activation of beta-catenin signaling.

Adenoviridae↗

PubMed2006

Runx2-mediated regulation of the zinc finger Osterix/Sp7 gene.

The zinc finger transcription factor Osterix (Osx) regulates bone formation and osteoblast differentiation in vitro and in vivo. We investigated the transcriptional mechanisms underlying the mouse Osx expression by isolating and characterizing its 5' upstream region. We performed 5' RACE on mRNA isolated from murine chondroprogenitor cells and determined a cap site of Osx approximately -99 nucleotides upstream of the initiation codon. Sequence analysis of this TATA-less promoter shows several putative response elements for Sox9, VDRE, Runx and Sp1. Transfection of the Osx promoter driving the luciferase reporter gene into C3H10T1/2 and ATDC5 cells shows a strong basal promoter activity between 565 bp and 2 kb. Deletion mutant analyses show that the most proximal 852 kb of the Osx promoter contains the highest activating domains, while strong repressive domains were identified between 1.8 and 2 kb. Over-expression experiments indicate that Runx2 significantly transactivates the Osx promoter by at least 2 fold indicating that Osx is downstream of Runx2 in mesenchymal cells. This up-regulation was abrogated when the Runx2 responsive element on the Osx promoter was mutated. Finally, we show that Runx2 specifically binds to this DNA element in the Osx promoter. Thus our results show for the first time Osx transcriptional regulation through the bone and cartilage related transcription factor Runx2.

Alternative Splicing↗

PubMed2006

Tumor necrosis factor-alpha increases circulating osteoclast precursor numbers by promoting their proliferation and differentiation in the bone marrow through up-regulation of c-Fms expression.

Osteoclasts are essential cells for bone erosion in inflammatory arthritis and are derived from cells in the myeloid lineage. Recently, we reported that tumor necrosis factor-alpha (TNFalpha) increases the blood osteoclast precursor (OCP) numbers in arthritic patients and animals, which are reduced by anti-TNF therapy, implying that circulating OCPs may have an important role in the pathogenesis of erosive arthritis. The aim of this study is to investigate the mechanism by which TNFalpha induces this increase in OCP frequency. We found that TNFalpha stimulated cell division and conversion of CD11b+/Gr-1-/lo/c-Fms- to CD11b+/Gr-1-/lo/c-Fms+ cells, which was not blocked by neutralizing macrophage colony-stimulating factor (M-CSF) antibody. Ex vivo analysis of monocytes demonstrated the following: (i) blood CD11b+/Gr-1-/lo but not CD11b-/Gr-1- cells give rise to osteoclasts when they were cultured with receptor activator NF-kappaB ligand and M-CSF; and (ii) TNF-transgenic mice have a significant increase in blood CD11b+/Gr-1-/lo cells and bone marrow proliferating CD11b+/Gr-1-/lo cells. Administration of TNFalpha to wild type mice induced bone marrow CD11b+/Gr-1-/lo cell proliferation, which was associated with an increase in CD11b+/Gr-1-/lo OCPs in the circulation. Thus, TNFalpha directly stimulates bone marrow OCP genesis by enhancing c-Fms expression. This results in progenitor cell proliferation and differentiation in response to M-CSF, leading to an enlargement of the marrow OCP pool. Increased marrow OCPs subsequently egress to the circulation, forming a basis for elevated OCP frequency. Therefore, the first step of TNF-induced osteoclastogenesis is at the level of OCP genesis in the bone marrow, which represents another layer of regulation to control erosive disease.

Animals↗

PubMed2006

Anti-RANKL therapy for inflammatory bone disorders: Mechanisms and potential clinical applications.

Focal bone loss around inflamed joints in patients with autoimmune disease, such as rheumatoid arthritis, remains a serious clinical problem. The recent elucidation of the RANK/RANK-ligand/OPG pathway and its role as the final effector of osteoclastogenesis and bone resorption has brought a tremendous understanding of the pathophysiology of inflammatory bone loss, and has heightened expectation of a novel intervention. Here, we review the etiology of inflammatory bone loss, the RANK/RANK-ligand/OPG pathway, and the clinical development of anti-RANK-ligand therapy.

Arthritis↗

PubMed2006

Wnt induction of chondrocyte hypertrophy through the Runx2 transcription factor.

We investigated the molecular mechanisms underlying canonical Wnt-mediated regulation of chondrocyte hypertrophy using chick upper sternal chondrocytes. Replication competent avian sarcoma (RCAS) viral over-expression of Wnt8c and Wnt9a, upregulated type X collagen (col10a1) and Runx2 mRNA expression thereby inducing chondrocyte hypertrophy. Wnt8c and Wnt9a strongly inhibited mRNA levels of Sox9 and type II collagen (col2a1). Wnt8c further enhanced canonical bone morphogenetic proteins (BMP-2)-induced expression of Runx2 and col10a1 while Wnt8c and Wnt9a inhibited TGF-beta-induced expression of Sox9 and col2a1. Over-expression of beta-catenin mimics the effect of Wnt8c and Wnt9a by upregulating Runx2, col10a1, and alkaline phosphatase (AP) mRNA levels while it inhibits col2a1 transcription. Western blot analysis shows that Wnt8c and beta-catenin also induces Runx2 protein levels in chondrocytes. Thus, our results indicate that activation of the canonical beta-catenin Wnt signaling pathway induces chondrocyte hypertrophy and maturation. We further investigated the effects of beta-catenin-TCF/Lef on Runx2 promoter. Co-transfection of lymphoid enhancer factor (Lef1) and beta-catenin in chicken upper sternal chondrocytes together with deletion constructs of the Runx2 promoter shows that the proximal region spanning the first 128 base pairs of this promoter is responsible for the Wnt-mediated induction of Runx2. Mutation of the TCF/Lef binding site in the -128 fragment of the Runx2 promoter resulted in loss of its responsiveness to beta-catenin. Additionally, gel-shift assay analyses determined the DNA/protein interaction of the TCF/Lef binding sites on the Runx2 promoter. Finally, our site-directed mutagenesis data demonstrated that the Runx2 site on type X collagen promoter is required for canonical Wnt induction of col10a1. Altogether we demonstrate that Wnt/beta-catenin signaling is regulated by TGF-beta and BMP-2 in chick upper sternal chondrocytes, and mediates chondrocyte hypertrophy at least partly through activation of Runx2 which in turn may induce col10a1 expression.

Alkaline Phosphatase↗

PubMed2006

Initial observations of reduced uroflow in transgenic adenocarcinoma of murine prostate.

OBJECTIVES: To characterize the voiding function in a transgenic adenocarcinoma of murine prostate (TRAMP) mouse of advanced age, because it might provide a useful model of slow-onset bladder outlet obstruction. Spontaneous death from urinary outlet obstruction occurs in the TRAMP mouse. METHODS: A metabolic cage without a fecal separation screen was placed above a precision balance that reported the mass of the excreta pan every 100 ms. A computational algorithm identified voids suitable for uroflow assessment from other excretory events. A series of images were obtained automatically before and during the excretory events. RESULTS: The TRAMP mouse displayed a pulsatile voiding pattern characterized by a reduction in uroflow, prolongation of voiding, and droplet formation at the tip of the prepuce. Postmortem histologic examination revealed gross enlargement of the prostate, a suburethral tumor, dilation of the lumen of the urethra, and proteinaceous debris in the urethra and bladder. CONCLUSIONS: The observed changes were consistent with urethral obstruction induced by prostate enlargement and/or suburethral tumor. Additional studies are required to ascertain whether prostate enlargement per se is sufficient to produce urethral obstruction in the TRAMP mouse. This transgenic mouse strain may provide a model of slow-onset outlet obstruction that is more representative of bladder outlet obstruction caused by benign prostatic hyperplasia than is the obstruction produced by urethral ligation.

Adenocarcinoma↗

PubMed2006

Periprosthetic osteolysis: an immunologist's update.

PURPOSE OF REVIEW: Inflammation-induced osteolysis is a problem in both inflammatory arthritis and total joint arthroplasty. New drug therapies have been shown to slow, halt, or even reverse the osteolysis associated with inflammatory arthritis. Unfortunately, similar advances in the medical treatment of periprosthetic osteolysis have not occurred. This review will update the state of periprosthetic osteolysis. RECENT FINDINGS: Preliminary results with phase I and II clinical trials with AMG-162, a human IgG2 that binds receptor activator of nuclear factor kappaB (RANK) ligand, have been reported. Based on these results AMG-162 appears to be safe and to have a potent effect on osteoclast function. Based on animal studies, it is expected that regents such as AMG-162 that block RANK-ligand/RANK interaction will have activity in inflammation-induced osteolysis. Volumetric three-dimensional and magnetic resonance imaging scans for detecting and quantifying periprosthetic osteolysis have been validated in cadaver studies. Lymphocytic infiltrates and positive skin tests to cobalt have been found in patients with periprosthetic osteolysis after second generation metal-on-metal prostheses. These findings again raise the question of whether metal allergy may contribute to implant failure in these patients. A new subset of T helper cells that are neither Th1 nor Th2, but secrete a unique pattern of cytokines including IL-17, has recently been discovered. The importance of these cells in modifying particle-induced osteolysis remains to be determined. SUMMARY: There have been significant advances in our understanding of periprosthetic osteolysis, imaging technology to quantify osteolysis, and drug development. The time now seems ripe to translate these advances in clinical trials.

Antibodies, Monoclonal↗

PubMed2006

Osteoclast precursors: cytokine-stimulated immunomodulators of inflammatory bone disease.

PURPOSE OF REVIEW: Proinflammatory cytokines produced in inflamed joints promote the generation and release from the bone marrow of osteoclast precursors (OCPs). These OCPs are recruited to inflamed joints where, in response to proinflammatory cytokines, they differentiate into osteoclasts and behave as immunomodulatory cells. We review the regulation of OCP formation and differentiation, and propose that they are not only sources of bone-resorbing osteoclasts, but also of immune cells that influence the progression of inflammatory bone diseases. RECENT FINDINGS: OCP numbers are increased in the blood of humans and animals with tumor necrosis factor (TNF)-mediated arthritis and they correlate with serum TNF levels, which can be lowered by anti-TNF therapy. TNF stimulates bone marrow OCP generation in part by increasing their expression of c-Fms. These precursors then leave the bone marrow and circulate in the blood stream. Receptor activator NF-kappaB ligand (RANKL) and TNF, which are elevated in inflamed joints, mediate OCP differentiation into osteoclasts and other cell types, including dendritic cells. They also stimulate OCPs to produce proinflammatory cytokines and chemokines. RANKL inhibition prevents joint inflammation in TNF-mediated arthritis. SUMMARY: OCPs are effector immunomodulatory cells in inflammatory bone diseases. They respond to and secrete cytokines, and are therefore targets for anti-inflammatory therapy.

Bone Diseases↗

PubMed2006

Autoimmunity and bone.

Focal erosions of cartilage and bone, which occur in the joints of patients with autoimmune inflammatory arthritis (i.e., rheumatoid arthritis (RA) and psoriatic arthritis [PsA]), represent the most debilitating and irreversible components of the disease. Over the last decade, seminal breakthroughs in our understanding of the cells and signal transduction pathways central to this process have been elucidated. From this information an established paradigm has been developed to explain focal erosions in which osteoclasts responsible for erosions are derived from bone marrow-derived myeloid precursors. Using the tumor necrosis factor (TNF) transgenic mouse model of erosive arthritis and anti-TNF clinical trials with PsA patients, we have demonstrated that systemic TNF induces the migration of CD11b+ osteoclast precursors (OCP) from the bone marrow into peripheral blood. These OCP can then enter the joints in blood vessels, translocate across the receptor activator of NF-kappaB ligand (RANKL) rich inflamed synovium, and differentiate into active osteoclasts. In direct contrast to this, systemic lupus erythematosus (SLE) patients appear to have an innate resistance to bone resorption. Our hypothesis to explain this phenomenon is that systemic interferon-alpha (IFN-alpha) diverts the bone marrow-derived myeloid precursors away from the osteoclast lineage and stimulates their differentiation into dendritic cells (DC). In support of this model, several labs have used microarray analyses to define the IFN-induced transcriptome in peripheral blood mononuclear cells (PBMC) from SLE patients. Here we propose the hypothesis that systemic TNF induces osteoclastic differentiation of PBMC in PsA patients that correlates with their erosive disease, and that the innate immune TNF/IFN axis in patients with autoimmune disease dictates their erosive phenotype. To demonstrate this, we injected wild-type C57B/6 and TNF-Tg mice with poly I:C, which is known to induce systemic IFN responses, and show its dominant effects on increasing the number of circulating CD11b+/CD11c+ precursor dendritic cells (pDC), concomitant with a dramatic reduction in CD11b+/CD11c- OCP. Thus, systemic factors produced by autoimmunity have a dramatic impact on active myelopoiesis and bone homeostasis.

Animals↗

PubMed2006

Safety and efficacy of ultraviolet-a light-activated gene transduction for gene therapy of articular cartilage defects.

BACKGROUND: Gene therapies for articular cartilage defects are limited by the absence of an in vivo delivery system that can mediate site-specific transduction restricted to within the margins of the defect during routine arthroscopy. We have proposed the use of ultraviolet light to stimulate gene expression following infection by recombinant adeno-associated virus (rAAV). However, research has demonstrated that short-wavelength ultraviolet light (ultraviolet C), while effective, is neither safe nor practical for this purpose. We evaluated the safety and efficacy of long-wavelength ultraviolet light (ultraviolet A) from a laser to induce light-activated gene transduction in articular chondrocytes in vitro and in vivo. METHODS: The effects of ultraviolet A from a 325-nm helium-cadmium laser, delivered through a fiberoptic cable, on cytotoxicity, mutagenesis, intracellular reactive oxygen species, and light-activated gene transduction of human articular chondrocytes were evaluated in dose-response experiments of primary cultures. Cytotoxicity was determined by trypan blue exclusion. The presence of pyrimidine dimers in purified genomic DNA was determined by enzyme-linked immunosorbent assays. Intracellular reactive oxygen species levels were determined by flow cytometry at one hour and twenty-four hours. In vitro light-activated gene transduction with rAAV vectors expressing the green fluorescent protein (eGFP) or beta-galactosidase (LacZ) was determined by fluorescence microscopy and bioluminescence assays, respectively. In vivo light-activated gene transduction was quantified by stereotactic immunohistochemistry for beta-galactosidase in rabbit articular cartilage defects in the patellar groove that had been irradiated with +/-6000 J/m2 of ultraviolet A one week after direct injection of 10(7) transducing units of rAAV-eGFP. RESULTS: Ultraviolet A failed to induce significant cytotoxicity at all fluencies below 6000 J/m2. Dose-dependent cytotoxicity was observed at greater fluencies. In contrast to ultraviolet C, which induced significant (p < 0.05) pyrimidine dimer formation at all fluencies in a dose-dependent manner, ultraviolet A failed to induce DNA modifications. Conversely, ultraviolet C proved to be a poor inducer of intracellular reactive oxygen species, while ultraviolet A immediately induced high levels of intracellular reactive oxygen species, which were completely resolved twenty-four hours later. Ultraviolet A demonstrated significant light-activated gene transduction effects in vitro, which were dose-dependent (p < 0.05). In vivo, ultraviolet A mediated a tenfold increase in transduction in which 40.8% of the superficial chondrocytes adjacent to the defect stained positive for green fluorescent protein compared with 5.2% in the knees treated with no ultraviolet A (p < 0.006). CONCLUSIONS: These results provide what we believe is the first formal demonstration of an agent that can induce rAAV transduction in the complete absence of cytotoxicity and DNA modification. They also suggest that the mechanism by which long-wavelength ultraviolet light mediates site-specific gene expression is by means of the induction of intracellular reactive oxygen species. Finally, laser-derived ultraviolet A can be readily transferred through a fiberoptic cable to mediate light-activated gene transduction in vivo.

Cartilage, Articular↗

PubMed2005

Tumor necrosis factor promotes Runx2 degradation through up-regulation of Smurf1 and Smurf2 in osteoblasts.

Tumor necrosis factor (TNF) plays an important role in the pathogenesis of inflammatory bone loss through stimulation of osteoclastic bone resorption and inhibition of osteoblastic bone formation. Compared with the well established role of TNF in osteoclastogenesis, mechanisms by which TNF inhibits osteoblast function have not been fully determined. Runx2 is an osteoblast-specific transcription factor whose steady-state protein levels are regulated by proteasomal degradation, mediated by the E3 ubiquitin ligases, Smurf1 and Smurf2. We hypothesized that TNF inhibits osteoblast function through Smurf-mediated Runx2 degradation. We treated C2C12 and 2T3 osteoblast precursor cell lines and primary osteoblasts with TNF and found that TNF, but not interleukin-1, significantly increased Smurf1 and Smurf2 expression. TNF increased the degradation of endogenous or transfected Runx2 protein, which was blocked by treating cells with a proteasomal inhibitor or by infecting cells with small interfering (si)RNA against Smurf1 or Smurf2. TNF inhibited the expression of bone morphogenetic protein and transforming growth factor-beta signaling reporter constructs, and the inhibition of each was blocked by Smurf1 siRNA and Smurf2 siRNA, respectively. Overexpression of Smurf1 and/or Smurf2 siRNAs prevented the inhibitory effect of TNF on Runx2 reporter. Consistent with these in vitro findings, bones from TNF transgenic mice or TNF-injected wild type mice had increased Smurf1 and decreased Runx2 protein levels. We propose that one of the mechanisms by which TNF inhibits bone formation in inflammatory bone disorders is by promoting Runx2 proteasomal degradation through up-regulation of Smurf1 and Smurf2 expression.

Animals↗

PubMed2005

Increased radiation-induced apoptosis of Saos2 cells via inhibition of NFkappaB: a role for c-Jun N-terminal kinase.

To elucidate the possible effect of NFkappaB on radioresistance, we used the osteosarcoma cell line Saos2, stably expressing the NFkappaB constitutive inhibitor, mIkappaB (Saos2-mIkappaB) or stably transfected with the empty vector (Saos2-EV). Ionizing radiation induced "intrinsic" apoptosis in Saos2-mIkappaB cells but not in Saos2-EV control cells, with intact NFkappaB activity. We find as expected, that this NFkappaB activity was enhanced following irradiation in the Saos2-EV control cells. On the other hand, inhibition of NFkappaB signaling in Saos2-mIkappaB cells led to the upregulation of the pro-apoptotic systems, such as Bax protein and c-Jun N-terminal Kinase (JNK)/c-Jun/AP1 signaling. Inhibition of NFkappaB resulted in decreased expression of the DNA damage protein GADD45beta, a known inhibitor of JNK. Subsequently, JNK activation of c-Jun/AP-1 proteins increased radiation-induced apoptosis in these mutants. Radiation-induced apoptosis in Saos2-mIkappaB cells was inhibited by the JNK specific inhibitor SP600125 as well as by Bcl-2 over-expression. Furthermore, release of cytochrome-c from mitochondria was increased and caspase-9 and -3 were activated following irradiation in Saos2-mIkappaB cells. Antisense inhibition of GADD45beta in Saos2-EV cells significantly enhanced apoptosis following irradiation. Our results demonstrate that radioresistance of Saos2 osteosarcoma cells is due to NFkappaB-mediated inhibition of JNK. Our study brings new insight into the mechanisms underlying radiation-induced apoptosis of osteosarcoma, and may lead to development of new therapeutic strategies against osteosarcoma.

Antigens, Differentiation↗

PubMed2005

A genome-wide expression profile and system-level integration of nuclear factor kappa B regulated genes reveals fundamental metabolic adaptations during cell growth and survival.

A murine lung alveolar carcinoma cell line (WT-Line 1) and its equally tumorigenic but non-malignant derivative transduced with a dominant negative inhibitor of NF-kappaB (mI-kappaB-Line 1), were profiled on the Affymetrix 19000 gene array platform. Two differentially expressed gene clusters were identified and integrated into a functional model. The downregulation of anti-oxidant defenses, in mI-kappaB-Line 1 cells, correlates with high levels of reactive oxygen species (ROS) and ROS damage to cellular macromolecules while the upregulation of metabolic nuclear receptors correlates with an adaptive/survival response, which involves a shift in energy metabolism toward beta-oxidative respiration. Accordingly, mI-kappaB-Line 1 cells are markedly sensitized to pharmacologic inhibition of beta-oxidative respiration. These findings are indicative of compensatory changes that could undermine anti-cancer therapies targeting NF-kappaB.

Adaptation, Physiological↗

PubMed2005

Smad7 mediates inhibition of Saos2 osteosarcoma cell differentiation by NFkappaB.

The transcription factor NFkappaB is constitutively activated in various tumor cells where it promotes proliferation and represses apoptosis. The bone morphogenetic proteins (BMPs) delay cell proliferation and promote differentiation and apoptosis of bone cells through activation of Smad downstream effectors and via Smad-independent mechanisms. Thus, NFkappaB and BMP pathways play opposing roles in regulating osteoblastic cell fate. Here, we show that in osteosarcoma Saos2 osteoblasts, NFkappaB regulates the activity of the BMP/Smad signaling. Inhibition of NFkappaB by overexpression of mIkappaB leads to the induction of osteoblast differentiation. Saos2 cells overexpressing mIkappaB (Saos2-mIkappaB) exhibit higher expression of osteoblast phenotypic genes such as alkaline phosphatase, Runx2 and osteocalcin and are more responsive to BMP2 in comparison to wild-type cells (Saos2-wt) or empty vector infected controls (Saos2-EV). Furthermore, BMP-2 signaling and Smad phosphorylation are significantly increased in Saos2-mIkappaB cells in comparison to Saos2-EV cells. Inhibition of NFkappaB signaling in Saos2-mIkappaB cells is associated with decreased expression of the BMP signaling inhibitor Smad7. While gain of Smad7 function in Saos2-mIkappaB cells results in inhibition of BMP signaling, anti-sense knockdown of Smad7 in Saos2-EV cells leads to upregulation of BMP signaling. We therefore conclude that in osteosarcoma Saos2 cells, NFkappaB represses BMP/Smad signaling and BMP2-induced differentiation through Smad7.

Alkaline Phosphatase↗

PubMed2005

The 3rd International Meeting on Gene Therapy in Rheumatology and Orthopaedics.

The 3rd International Meeting on Gene Therapy in Rheumatology and Orthopaedics was held in Boston, Massachusetts, USA in May 2004. Keystone lectures delivered by Drs Joseph Glorioso and Inder Verma provided comprehensive, up-to-date information on all major virus vectors. Other invited speakers covered the application of gene therapy to treatment of arthritis, including the latest clinical trial in rheumatoid arthritis, as well as lupus and Sjögren's syndrome. Applications in mesenchymal stem cell biology, tissue repair, and regenerative medicine were also addressed. The field has advanced considerably since the previous meeting in this series, and further clinical trials seem likely.

Genetic Therapy↗

PubMed2005

Gene therapy with human osteoprotegerin decreases callus remodeling with limited effects on biomechanical properties.

Osteoprotegerin (OPG) is a naturally occurring protein, which prevents bone resorption by inhibition of osteoclastogenesis, function, and survival. Therefore, recombinant OPG may be an attractive drug in the treatment of chronic bone resorptive diseases such as osteoporosis. Gene therapy has the potential to achieve long-term treatment by delivering genes of anti-resorptive proteins to the recipient. The effects of OPG gene therapy on fracture healing have not been described previously. The influence of OPG gene therapy on callus formation, callus tissue structural strength, apparent material properties, and histology of tibia fractures in rats was investigated after 3 weeks and 8 weeks of healing. Intramuscular administration of adeno-associated virus (AAV) vector-mediated OPG resulted in increased levels of OPG in serum of approximately 100 ng/ml throughout the study period. Control animals with fractures received transduction with an AAV reporter gene construct (AAV-enhanced green fluorescent protein (eGFP)), and in this group serum OPG levels remained at baseline (<10 ng/ml). After 3 weeks of healing, AAV-OPG treatment reduced the number of osteoclasts in the callus tissue (33%, P < 0.001). However, AAV-OPG treatment did not influence callus dimensions, callus bone mineral content (BMC), fracture structural strength, or apparent callus tissue material properties. After 8 weeks of healing, AAV-OPG treatment reduced the number of osteoclasts in the callus tissue (31%, P < 0.001) compared with AAV-eGFP fractures. Furthermore, deposition of new woven bone at the fracture line of the original cortical bone was hampered (new woven bone present: in all AAV-eGFP animals, in 41% of AAV-OPG-treated animals, P < 0.001). AAV-OPG treatment also increased callus BMC (18%, P = 0.023) compared with AAV-eGFP fractures. AAV-OPG did not influence callus dimensions, structural strength of the fractures, or ultimate stress, whereas elastic modulus was reduced in the AAV-OPG groups (37%, P = 0.039). The experiment demonstrates that AAV-OPG gene therapy decreases the fracture remodeling, but this does not influence the structural strength of healing fractures.

Animals↗

PubMed2005

Smad3-deficient chondrocytes have enhanced BMP signaling and accelerated differentiation.

UNLABELLED: Smad3 deficiency accelerates chondrocyte maturation and leads to osteoarthritis. Primary chondrocytes without Smad3 lack compensatory increases of TGF-beta signaling factors, but BMP-related gene expression is increased. Smad2 or Smad3 overexpression and BMP blockade abrogate accelerated maturation in Smad3-/- chondrocytes. BMP signaling is increased in TGF-beta deficiency and is required for accelerated chondrocyte maturation. INTRODUCTION: Disruption of TGF-beta signaling results in accelerated chondrocyte maturation and leads to postnatal dwarfism and premature osteoarthritis. The mechanisms involved in this process were studied using in vitro murine chondrocyte cultures. MATERIALS AND METHODS: Primary chondrocytes were isolated from the sterna of neonatal wildtype and Smad3-/- mice. Expressions of maturational markers, as well as genes involved in TGF-beta and BMP signaling were examined. Chondrocytes were treated with TGF-beta and BMP-2, and effects on maturation-related genes and BMP/TGF-beta responsive reporters were examined. Recombinant noggin or retroviral vectors expressing Smad2 or Smad3 were added to the cultures. RESULTS: Expression of colX and other maturational markers was markedly increased in Smad3-/- chondrocytes. Smad3-/- chondrocytes lacked compensatory increases in Smad2, Smad4, TGFRII, Sno, or Smurf2 and had reduced expression of TGF-beta1 and TGFRI. In contrast, Smad1, Smad5, BMP2, and BMP6 expression was increased, suggesting a shift from TGF-beta toward BMP signaling. In Smad3-/- chondrocytes, alternative TGF-beta signaling pathways remained responsive, as shown by luciferase assays. These non-Smad3-dependent TGF-beta pathways reduced colX expression and alkaline phosphatase activity in TGF-beta-treated Smad3-/- cultures, but only partially. In contrast, Smad3-/- chondrocytes were more responsive to BMP-2 treatment and had increased colX expression, phosphoSmads 1, 5, and 8 levels, and luciferase reporter activity. Overexpression of both Smad2 and Smad3 blocked spontaneous maturation in Smad3-deficient chondrocytes. Maturation was also abrogated by the addition of noggin, an extracellular BMP inhibitor. CONCLUSIONS: These findings show a key role for BMP signaling during the chondrocyte maturation, occurring with loss of TGF-beta signaling with important implications for osteoarthritis and cartilage diseases.

Animals↗