Enhancing osteogenic capabilities of bone matrix for the treatment of fractures and segmental defects using growth factors is an active area of research. Recently, synthetic peptides like AC- 100, TP508 or p-15 corresponding to biologically active sequences of matrix proteins have been proven to stimulate bone formation. The platelet-derived growth factor (PDGF) BB has been identified as an important paracrine factor in early bone healing. We hypothesized that the combined use of PDGF-BB with synthetic peptides could result in an increase in proliferation and calcification of osteoblast-like cells. Osteoblast-like cell cultures were treated with PDGF and synthetic peptides, singly and as combinations, and compared to non-treated control cell cultures. The cultures were evaluated at days 2, 5, and 10 in terms of cell proliferation, calcification and gene expression of alkaline phosphate, collagen I and osteocalcin. Experimental findings revealed that the addition of PDGF, p-15 and TP508 and combinations of PDGF/AC-100, PDGF/p-15 and PDGF/TP508 resulted in an increase in proliferating osteoblasts, especially in the first 5 days of cultivation. Proliferation did not significantly differ between single factors and factor combinations (p > 0.05). The onset of calcification in osteoblasts occurred earlier and was more distinct compared to the corresponding control or PDGF stimulation alone. Significant difference was found for the combined use of PDGF/p-15 and PDGF/AC-100 (p < 0.05). Our findings indicate that PDGF exhibits cooperative effects with synthetic peptides in differentiation and proliferation. These cooperative effects cause a significant early calcification of osteoblast-like cells (p < 0.05). We suggest the combination of synthetic peptides and PDGF as a potential clinical approach for accelerating bone healing or coating osteosynthesis materials.
Abstract Background An altered susceptibility of lung fibroblasts to Fas-induced apoptosis has been implicated in the pathogenesis of pulmonary fibrosis; however, the underlying mechanism is not completely understood. Here, we studied the susceptibility of lung fibroblasts, obtained from patients with (f-fibs) and without pulmonary fibrosis (n-fibs), to FasL- (CD95L/APO-1) induced apoptosis in relation to the expression and the amounts of membrane-bound and soluble Fas. We also analysed the effects of tumor necrosis factor-β on FasL-induced cell death. Methods Apoptosis was induced with recombinant human FasL, with and without prior stimulation of the fibroblasts with tumor necrosis factor-α and measured by a histone fragmentation assay and flow cytometry. The expression of Fas mRNA was determined by quantitative PCR. The expression of cell surface Fas was determined by flow cytometry, and that of soluble Fas (sFas) was determined by enzyme-linked immunosorbent assay. Results When compared to n-fibs, f-fibs were resistant to FasL-induced apoptosis, despite significantly higher levels of Fas mRNA. F-fibs showed lower expression of surface-bound Fas but higher levels of sFas. While TNF-α increased the susceptibility to FasL-induced apoptosis in n-fibs, it had no pro-apoptotic effect in f-fibs. Conclusions The data suggest that lower expression of surface Fas, but higher levels of apoptosis-inhibiting sFas, contribute to the resistance of fibroblasts in lung fibrosis against apoptosis, to increased cellularity and also to increased formation and deposition of extracellular matrix.
Apoptosis is a principal mechanism in metazoans by which superfluous or potentially harmful cells are eliminated.Deregulation of this process leads to a variety of diseases such as cancer and autoimmune diseases.Stimuli that can induce apoptosis are relatively diverse, and include the death factors (Fas ligand, tumor necrosis factor and TRAIL), DNA damage, and oxidative stress.Regardless of the origin of the apoptotic stimulus, commitment to apoptosis leads to activation of caspases, a family of cysteine proteases.Cleavage of a select group of cellular substrates by caspases is responsible for the morphological and biochemical changes that characterize apoptotic cell death.The degradation of nuclear DNA into nucleosomal units is one of the features of apoptotic cell death, and is mediated by a caspase-activated DNase (CAD).Cells deficient in CAD undergo cell death without the DNA fragmentation, but CAD-null mice did not show any adverse phenotypes.A close examination of the apoptotic cells in these mice indicated that apoptotic cells are always in macrophages.It seems that at an early stage of apoptosis, the dying cells present an 'eat me signal' on their surface.This signal is recognized by macrophages for engulfment, and DNase II in the lysosomes of macrophages degrades DNA of apoptotic cells.Mice deficient in both CAD and DNase II genes were established, and the development of various organs was found to be severely impaired in these mutant mice.The mice accumulated a large amount of undigested DNA in macrophages in various tissues during development.This accumulation of DNA in macrophages activated the innate immunity to induce the expression of the interferon β gene.The interferon thus produced seems to be responsible for the impaired tissue development.These results indicate that the degradation of DNA during apoptotic cell death is an essential step of apoptosis to maintain mammalian homeostasis.
The aim of the present study was to investigate the expression of Fas in periarticular tenocytes of patients with osteoarthritis (OA) and to study their susceptibility to Fas ligand-mediated apoptosis. Tendon samples were obtained from the quadriceps femoris muscle of patients with knee OA and used for histological evaluation, for immunohistochemical detection of Fas, and to establish tenocyte cultures. The expression of Fas mRNA was determined by quantitative PCR. Levels of soluble Fas and soluble tumour necrosis factor (TNF) receptor I were measured using ELISA. Apoptosis was induced with recombinant human Fas ligand and measured by a histone fragmentation assay and flow cytometry. The effects of TNF-alpha were studied by stimulation with TNF-alpha alone or 24 hours before the induction of apoptosis. Tendon samples from non-OA patients were used as controls. Histological evaluation revealed degenerative changes in the tendons of all OA patients but not in the controls. Fas was detected by immunohistochemistry in all specimens, but quantitative PCR revealed significantly higher levels of Fas mRNA in OA tenocytes. In contrast, lower levels of soluble Fas were found in OA tenocytes by ELISA. OA tenocytes were significantly more susceptible to Fas ligand induced apoptosis than were control cells. TNF-alpha reduced the Fas ligand induced apoptosis in OA tenocytes but had no effects on control tenocytes. These data suggest that knee OA is associated with higher susceptibility of periarticular tenocytes to Fas ligand induced apoptosis because of higher expression of Fas but lower levels of apoptosis-inhibiting soluble Fas. These changes may contribute to decreased cellularity in degenerative tendons and promote their rupturing. The antiapoptotic effects of TNF-alpha in OA tenocytes most likely reflect regenerative attempts and must be taken into account when anti-TNF strategies are considered for OA.
The severe bone destruction during rheumatoid arthritis (RA) is mainly mediated by osteoclasts and thus, reducing osteoclast number is a keystep in ameliorating the outcome of the disease. Yersinia strains are secreting a broad number of effector proteins. The yersinia outer protein M (YopM) has the ability to translocate into host cells by two N-terminal alpha-helices and subsequently down-regulates inflammatory response in infected host tissues.
Objectives
We investigated the effect of recombinant YopM on osteoclastogenesis in vitro and bone destruction in the hTNFtg mouse model in vivo.
Methods
After cloning recombinant YopM into pET-24 b (+) expression vector, it was expressed in Bl21 cells and purified. The uptake of YopM into bone marrow macrophages (BMMs) and osteoclasts was analysed using Cy3-coupled YopM and laser scanning microscopy. The effect of YopM on osteoclastogenesis in an in vitro osteoclast formation assay was studied. The effects of YopM on the activation of ERK, p-38, AKT and NFkappaB signalling was determined by Western Blot analysis. With respect to a potential in vivo application of YopM, we analysed the distribution of YopM-Cy5 injected in the hindpaws of hTNFtg mice by fluorescence reflection imaging (FRI). Finally, we treated hTNFtg mice with YopM and an inactive deletion mutant of YopM. Clinical parameters were recorded and histomorphometric analyses of joint destruction were performed in hind paw sections.
Results
In contrast to mature osteoclasts, YopM penetrated the cell membrane of BMMs in vitro and accumulated in the cells. The incubation of BMMs with YopM resulted in a strong reduction of osteoclast precursors and mature osteoclasts. We found that YopM reduced the activation of NF-kB by reducing the phosphorylation of IkB alpha. Phosphorylation of MAP kinases and AKT, however, was not altered by YopM. YopM-Cy5 injected into the hind paws was detectable by FRI during the monitored time of 48 houres. The treatment of hTNFtg mice with YopM showed a delayed onset of paw swelling and reduced bone destruction and inflammation in YopM treated hTNFtg mice in comparison to untreated hTNFtg mice.
Conclusions
These results suggest that YopM has the potential to reduce inflammation and bone destruction in vivo. Therefore, the underlying mechanisms and functional properties of YopM may serve as basis for the development of novel strategies for the treatment of RA.
Heterogeneity and multifactoriality complicate diagnostics and our understanding of pathogenesis of rheumatoid arthritis (RA).The only accepted serologic parameter (rheumatoid factor [RF]) is not disease specific, nor are any of several novel RA autoantibodies.We aimed at identifying profiles instead of individual autoreactivities allowing for unambiguous prediction of RA.Selected RA autoantigens were tested by ELISA (RF and anti-cyclic citrullinated peptide [anti-CCP]) or Western blot (heavy-chain-binding protein [BiP], heterogeneous ribonucleoprotein particle A2 [RA33/ hnRNP A2], calpastatin and calreticulin).Antibody reactivities were assayed from serum samples of 149 RA patients and 132 patients with other rheumatic diseases and from synovial fluids (SF) (58 RA, 65 non-RA).No single autoreactivity was sufficient for unambiguous prediction of RA.Frequencies of multiparameter profiles consisting of 3, 4, 5 and 6 autoreactivites were determined.Fifteen six-parameter serum profiles were exclusively expressed in RA patients, representing a cumulative sensitivity of 59%.Twelve SF profiles were exclusively expressed in 64% of RA patients.The self-learning classification algorithm CLASSIF1 was capable of accurately predicting RA when these profiles were present.Data profile analysis of RF/CCP/BiP/calpastatin/calreticulin/RA33 provided specific discrimination of 64% of RA.Most importantly, RA specific profiles were observed in 64% of patients with early disease (<12 months).For the first time, the accurate prediction of the class RA has been achieved by the use of multiparametric autoreactivity profiles.Because of early expression in disease, these profiles make it possible to start a disease-modifying therapy long before irreversible bone and joint destruction may develop.Additional RA-specific profiles are required to cover the entire group of RA patients.
Based on our previous data that the heparan sulfate proteoglycan syndecan-4 (Sdc4) is involved in cartilage breakdown during osteoarthritis, the authors analysed the distribution and functional role of syndecan-4 during endochondral ossification. The authors investigated this process during limb development in mouse embryogenesis and studied its contribution in fracture healing in adult bones, a process that in some respect recapitulates the sequence of biological events of endochondral ossification during skeletal development.
Methods
The authors analysed syndecan-4 promoter activity in mouse embryos (E12- 17) by staining for β-galactosidase in sdc4−/−lacZ knock-in mice. For functional analysis, the authors assessed bone development in wild type and sdc4−/− animals by alcian blue/alizarin red staining and compared the expression of proteins implicated in cell proliferation and matrix remodelling (PCNA, ADAMTS-4, aggrecan neoepitopes) by immunohistochemistry. Fracture healing experiments were performed using 12-week-old female sdc4−/− and wild type mice and induced standardised, stabilised femur shaft fractures. Fractured and native femurs were dissected for biomechanical testing and maximum torque, angle at max. torque were determined, torsional stiffness was calculated. After 7, 14 and 28 days femurs were decalcified and embedded in paraffin. After alcian blue and Masson Goldner stainings, ratio of cartilage area and bone area to total callus area were measured.
Results
At E12.0 a strong activity of the syndecan-4 promoter occurred at sites of cartilage condensations. In later stages syndecan-4 was detected in the growth plates of long bones. On the cellular level, syndecan-4 expression was detectable mainly in proliferative and hypertrophic chondrocytes. When the authors compared endochondral ossification in wt and sdc4−/− mice, they found the loss of syndecan-4 was associated with a marked inhibition of chondrocyte proliferation and a slight inhibition in the mineralisation of appendicular bones. This was accompanied by a loss of aggrecanase expression and a significantly reduced staining for ADAMTS generated aggrecan neoepitops in the epiphysial cartilage in E16.5 tibiae of sdc-4 KO animals. In line with these data, histomorphometric analysis of fractured femurs from sdc4−/− mice demonstrated increased callus and cartilage formation compared to wild type mice in the early stage of fracture healing.
Conclusions
Our data demonstrate that syndecan-4 is critically involved in chondrocyte differentiation during endochondral ossification. Loss of syndecan-4 affects proliferation and matrix remodelling by aggrecanases. These findings may be of relevance mainly during fracture healing in adult bones supporting existing evidence that syndecan-4 plays an important role in metabolic events under inflammatory conditions.
