Treponema denticola increases MMP-2 expression and activation in the periodontium via reversible DNA and histone modifications.

Host-derived matrix metalloproteinases (MMPs) and bacterial proteases mediate destruction of extracellular matrices and supporting alveolar bone in periodontitis. The Treponema denticola dentilisin protease induces MMP-2 expression and activation in periodontal ligament (PDL) cells, and dentilisin-mediated activation of pro-MMP-2 is required for cellular fibronectin degradation. Here, we report that T. denticola regulates MMP-2 expression through epigenetic modifications in the periodontium. PDL cells were treated with epigenetic enzyme inhibitors before or after T. denticola challenge. Fibronectin fragmentation, MMP-2 expression, and activation were assessed by immunoblot, zymography, and qRT-PCR, respectively. Chromatin modification enzyme expression in T. denticola-challenged PDL cells and periodontal tissues were evaluated using gene arrays. Several classes of epigenetic enzymes showed significant alterations in transcription in diseased tissue and T. denticola-challenged PDL cells. T. denticola-mediated MMP-2 expression and activation were significantly reduced in PDL cells treated with inhibitors of aurora kinases and histone deacetylases. In contrast, DNA methyltransferase inhibitors had little effect, and inhibitors of histone acetyltransferases, methyltransferases, and demethylases exacerbated T. denticola-mediated MMP-2 expression and activation. Chronic epigenetic changes in periodontal tissues mediated by T. denticola or other oral microbes may contribute to the limited success of conventional treatment of chronic periodontitis and may be amenable to therapeutic reversal.

Quantification of human angiogenesis in immunodeficient mice using a photon counting-based method.

Testing new antiangiogenic drugs for cancer treatment requires the use of animal models, since stromal cells and extracellular matrices mediate signals to endothelial cells that cannot be fully reproduced in vitro. Most methods used for analysis of antiangiogenic drugs in vivo utilized histologic examination of tissue specimens, which often requires large sample sizes to obtain reliable quantitative data. Furthermore, these assays rely on the analysis of murine vasculature that may not be correlated with the responses of human endothelial cells. Here, we engineered human blood vessels in immunodeficient mice with human endothelial cells expressing luciferase, demonstrated that these cells line functional blood vessels, and quantified angiogenesis over time using a photon counting-based method. In a proof-of-principle experiment with PTK/ZK, a small molecule inhibitor of vascular endothelial growth factor (VEGF) tyrosine kinase receptors, a strong correlation was observed between the decrease in bioluminescence (9.12-fold) in treated mice and the actual decrease in microvessel density (9.16-fold) measured after retrieval of the scaffolds and immunohistochemical staining of endothelial cells. The method described here allows for quantitative and noninvasive investigation into the effects of anti-cancer drugs on human angiogenesis in a murine host.

Matrix and TGF-beta-related gene expression during human dental pulp stem cell (DPSC) mineralization.

We have recently reported the induction of dental pulp stem cells (DPSCs) into dentin-secreting odontoblast-like cells after stimulation by isolated dentin matrix components, thus mimicking the nature of tissue regeneration seen after tooth disease and injury. After confluency, the cells were further cultured for 21 d in the 10% fetal bovine serum (FBS) Dulbecco's modified Eagle's medium (DMEM) (control), and in this medium, with the addition of dentin extract (DE) and the mineralization supplement (MS) of ascorbic acid and beta-glycerophosphate (treatment). To identify genes associated with this process, specimens were analyzed with a HG-U133A human gene chip and Arrayassist software. A total of 425 genes, among them 21 matrix and eight TGF-beta-related genes, were either up- or downregulated in the experimental group in which the cells showed odontoblast-like differentiation and mineralization. Expression of selected genes was further confirmed by real-time polymerase chain reaction (PCR) analysis. Of the extracellular matrix (ECM)-related genes, two types of collagen genes were upregulated and seven others downregulated. Other ECM-related genes, for example fibulin-1, tenascin C, and particularly thrombospondin 1, were upregulated, and fibulin-2 was downregulated. Most noticeably, the matrix metalloproteinase 1 was induced by the treatment. In the TGF-beta superfamily, upregulation of the type II receptor, endoglin, and growth/differentiation factor 5 was coordinated with the downregulation of activin A, TGF-beta2, and TGF-beta1 itself. This study identifies the matrix and TGF-beta-related gene profiles during the DPSC cell mineralization in which several genes are reported for the first time to be associated with this process, thus greatly expanding our molecular knowledge of the induced disease repair process.

Bcl-2 acts in a proangiogenic signaling pathway through nuclear factor-kappaB and CXC chemokines.

Vascular endothelial growth factor (VEGF) induces expression of Bcl-2 in tumor-associated microvascular endothelial cells. We have previously reported that up-regulated Bcl-2 expression in microvascular endothelial cells is sufficient to enhance intratumoral angiogenesis and to accelerate tumor growth. We initially attributed these results to Bcl-2-mediated endothelial cell survival. However, in recent experiments, we observed that conditioned medium from Bcl-2-transduced human dermal microvascular endothelial cells (HDMEC-Bcl-2) is sufficient to induce potent neovascularization in the rat corneal assay, whereas conditioned medium from empty vector controls (HDMEC-LXSN) does not induce angiogenesis. These results cannot be attributed to the role of Bcl-2 in cell survival. To understand this unexpected observation, we did gene expression arrays that revealed that the expression of the proangiogenic chemokines interleukin-8 (CXCL8) and growth-related oncogene-alpha (CXCL1) is significantly higher in HDMEC exposed to VEGF and in HDMEC-Bcl-2 than in controls. Inhibition of Bcl-2 expression with small interfering RNA-Bcl-2, or the inhibition of Bcl-2 function with small molecule inhibitor BL-193, down-regulated CXCL8 and CXCL1 expression and caused marked decrease in the angiogenic potential of endothelial cells without affecting cell viability. Nuclear factor-kappaB (NF-kappaB) is highly activated in HDMEC exposed to VEGF and HDMEC-Bcl-2 cells, and genetic and chemical approaches to block the activity of NF-kappaB down-regulated CXCL8 and CXCL1 expression levels. These results reveal a novel function for Bcl-2 as a proangiogenic signaling molecule and suggest a role for this pathway in tumor angiogenesis.

In vitro differentiation and mineralization of human dental pulp cells induced by dentin extract.

In this study, the progenitor cells isolated from the human dental pulp were used to study the effects of ethylenediaminetetraacetic acid-soluble dentin extract (DE) on their differentiation and mineralization to better understand tissue injury and repair in the tooth. Mineralization of the matrix was increasingly evident at 14, 21, and 28 d after treatment with a mineralization supplement (MS) (ascorbic acid [AA], beta-glycerophosphate [beta-GP]) and MS + DE. Real-time polymerase chain reaction results showed type I collagen upregulation after the addition of MS + DE at 7 d. Alkaline phosphatase was downregulated after the mineralization became obvious at 14 d. Bone sialoprotein was shown to be upregulated in the mineralized cell groups at all time points and dentin sialophosphoprotein after 7 d. Core binding factor a 1 was upregulated by the treatment of MS and DE at 7, 14, and 21 d. These results indicated that the MS of AA, beta-GP, and DE synergistically induced cell differentiation of pulp progenitor cells into odontoblast-like cells and induced in vitro mineralization.

Recruitment of mesenchymal stem cells into prostate tumours promotes metastasis.

Tumours recruit mesenchymal stem cells to facilitate healing, which induces their conversion into cancer-associated fibroblasts that facilitate metastasis. However, this process is poorly understood on the molecular level. Here we show that CXCL16, a ligand for CXCR6, facilitates mesenchymal stem cell or very small embryonic-like cells recruitment into prostate tumours. CXCR6 signalling stimulates the conversion of mesenchymal stem cells into cancer-associated fibroblasts, which secrete stromal-derived factor-1, also known as CXCL12. CXCL12 expressed by cancer-associated fibroblasts then binds to CXCR4 on tumour cells and induces an epithelial-to-mesenchymal transition, which ultimately promotes metastasis to secondary tumour sites. Our results provide the molecular basis for mesenchymal stem cell recruitment into tumours and how this process leads to tumour metastasis.

In vitro differentiation and mineralization of dental pulp stem cells on enamel-like fluorapatite surfaces.

Our previous studies have shown good biocompatibility of fluorapatite (FA) crystal surfaces in providing a favorable environment for functional cell-matrix interactions of human dental pulp stem cells (DPSCs) and also in supporting their long-term growth. The aim of the current study was to further investigate whether this enamel-like surface can support the differentiation and mineralization of DPSCs, and, therefore, act as a potential model for studying the enamel/dentin interface and, perhaps, dentine/pulp regeneration in tooth tissue engineering. The human pathway-focused osteogenesis polymerase chain reaction (PCR) array demonstrated that the expression of osteogenesis-related genes of human DPSCs was increased on FA surfaces compared with that on etched stainless steel (SSE). Consistent with the PCR array, FA promoted mineralization compared with the SSE surface with or without the addition of a mineralization promoting supplement (MS). This was confirmed by alkaline phosphatase (ALP) staining, Alizarin red staining, and tetracycline staining for mineral formation. In conclusion, FA crystal surfaces, especially ordered (OR) FA surfaces, which mimicked the physical architecture of enamel, provided a favorable extracellular matrix microenvironment for the cells. This resulted in the differentiation of human DPSCs and mineralized tissue formation, and, thus, demonstrated that it may be a promising biomimetic model for dentin-pulp tissue engineering.

The stimulation of adipose-derived stem cell differentiation and mineralization by ordered rod-like fluorapatite coatings.

In this study, the effect of ordered rod-like FA coatings of metal discs on adipose-derived stem cell (ASC)'s growth, differentiation and mineralization was studied in vitro; and their mineral inductive effects in vivo. After 3 and 7 days, the cell number on the metal surfaces was significantly higher than those on the ordered and disordered FA surfaces. However, after 4 weeks much greater amounts of mineral formation was induced on the two FA surfaces with and even without osteogenesis induction. The osteogenic profiles showed the up regulation of a set of pro-osteogenic transcripts and bone mineralization phenotypic markers when the ASCs were grown on FA surfaces compared to metal surfaces at 7 and 21 days. In addition to BMP and TGFß signaling pathways, EGF and FGF pathways also appeared to be involved in ASC differentiation and mineralization. In vivo studies showed accelerated and enhanced mineralized tissue formation integrated within ordered FA coatings. After 5 weeks, over 80% of the ordered FA coating was integrated with a mineralized tissue layer covering the implants. Both the intrinsic properties of the FA crystals and the topography of the FA coating appeared to dominate the cell differentiation and mineralization process.

The effect of novel fluorapatite surfaces on osteoblast-like cell adhesion, growth, and mineralization.

There is increasing demand for biomedical implants to correct skeletal defects caused by trauma, disease, or genetic disorder. In this study, the MG-63 cells were grown on metals coated with ordered and disordered fluorapatite (FA) crystal surfaces to study the biocompatibility, initial cellular response, and the underlying mechanisms during this process. The long-term growth and mineralization of the cells were also investigated. After 3 days, the cell numbers on etched metal surface are significantly higher than those on the ordered and disordered FA surfaces, but the initial adherence of a greater number of cells did not lead to earlier mineral formation at the cell-implant interface. Of the 84 cell adhesion and matrix-focused pathway genes, an up- or down-regulation of a total of 15 genes such as integrin molecules, integrin alpha M and integrin alpha 7 and 8 was noted, suggesting a modulating effect on these adhesion molecules by the ordered FA surface compared with the disordered. Osteocalcin expression and the mineral nodule formation are most evident on the FA surfaces after osteogenic induction (OI) for 7 weeks. The binding of the ordered FA surfaces to the metal, with and without OI, was significantly higher than that of the disordered FA surfaces with OI. Most significantly, even without the OI supplement, the MG-63 cells grown on FA crystal surfaces start to differentiate and mineralize, suggesting that the FA crystal could be a simple and bioactive implant coating material.

A dominant function of IKK/NF-kappaB signaling in global lipopolysaccharide-induced gene expression.

Porphyromonas gingivalis is an etiologic pathogen of periodontitis that is one of the most common inflammatory diseases. Recently, we found that P. gingivalis LPS activated the transcription factor nuclear factor-kappaB (NF-kappaB) through the IkappaB kinase complex (IKK). NF-kappaB is a transcription factor that controls inflammation and host responses. In this study, we examined the role of IKK/NF-kappaBin P. gingivalis LPS-induced gene expression on a genome-wide basis using a combination of microarray and biochemical approaches. A total of 88 early response genes were found to be induced by P. gingivalis LPS in a human THP.1 monocytic cell lines. Interestingly, the induction of most of these genes was abolished or attenuated under the inactivation of IKK/NF-kappaB. Among those IKK/NF-kappaB-dependent genes, 20 genes were NF-kappaB-inducible genes reported previously, and 59 genes represented putative novel NF-kappaB target genes. Using transcription factor binding analysis, we found that most of these putative NF-kappaB target genes contained one or multiple NF-kappaB-binding sites. Also, some transcription factor-binding motifs were overrepresented in the promoter of both known and putative NF-kappaB-dependent genes, indicating that these genes may be regulated in a similar fashion. Furthermore, we found that several transcription factors associated with metabolic and inflammatory responses, including nuclear receptors, activator of protein-1, and early growth responses, were induced by P. gingivalis LPS through IKK/NF-kappaB, indicating that IKK/NF-kappaB may utilize these transcription factors to mediate secondary responses. Taken together, our results demonstrate that IKK/NF-kappaB signaling plays a dominant role in P. gingivalis LPS-induced early response gene expression, suggesting that IKK/NF-kappaB is a therapeutic target for periodontitis.

Combinatorial gene therapy for bone regeneration: cooperative interactions between adenovirus vectors expressing bone morphogenetic proteins 2, 4, and 7.

Bone morphogenetic proteins (BMPs) have demonstrated effectiveness as bone regeneration agents whether delivered as recombinant proteins or via gene therapy. Current gene therapy approaches use vectors expressing single BMPs. In contrast, multiple BMPs are coordinately expressed during bone development and fracture healing. Furthermore, BMPs likely exist in vivo as heterodimeric molecules having enhanced biological activity. In the present study, we test the hypothesis that gene therapy-based bone regeneration can be enhanced by expressing combinations of BMPs. For in vitro studies, mesenchymal cell lines were transduced with individual adenoviruses containing BMP2, 4, or 7 cDNA under control of a CMV promoter (AdBMP2, 4, 7) or virus combinations. Significantly, combined transduction with AdBMP2 plus AdBMP7 or AdBMP4 plus AdBMP7 resulted in a synergistic stimulation of osteoblast differentiation. This synergy is best explained by formation of BMP2/7 and 4/7 heterodimers. To test in vivo biological activity, fibroblasts were transduced with specific virus combinations and implanted into C57BL6 mice. Consistent with in vitro results, strong synergy was observed using combined AdBMP2/BMP7 treatment, which induced twofold to threefold more bone than would be predicted based on the activity of individual AdBMPs. These studies show that dramatic enhancement of osteogenesis can be achieved using gene therapy to express specific combinations of interacting regenerative molecules.

Cell-to-cell contact is critical for the survival of hematopoietic progenitor cells on osteoblasts.

Osteoblasts constitute part of the stromal cell support system in marrow for hematopoiesis, however little is known as to how they interact with hematopoietic stem cells (HSCs). In vitro studies have demonstrated that the survival of HSCs in co-culture with osteoblasts requires intimate cell-to-cell contact. This suggests that the osteoblast-derived factor(s) that supports stem cell activities are produced in very small quantities, are rapidly turned over, may be membrane-anchored and/or require the engagement of cell-cell adhesion molecules that are yet to be determined. In the present report we found that the survival of hematopoietic progenitor cells on osteoblasts is dependent upon the engagement of VLA-4 (alpha4beta1) and VLA-5 (alpha5beta1) receptors using function blocking antibodies. Cell-to-cell contact is required to support progenitor activity, but can be replaced if receptor-ligand engagement of the VLA-4 and LFA-1 complexes is provided through the use of recombinant ligands (fibronectin, ICAM-1, VCAM-1). Moreover, once these receptors were engaged, conditioned medium derived from HSCs grown on osteoblast ligands supported significantly greater hematopoietic progenitors in vitro than did osteoblast-conditioned or HSC-conditioned medium alone. While the molecules present in the co-cultured medium remain to be identified, the data suggest that hematopoietic cells cooperate with osteoblasts to assemble the various marrow microenvironments by directing the synthesis of osteoblast-derived cytokines to improve HSC survival.

Identification of potential modifiers of Runx2/Cbfa1 activity in C2C12 cells in response to bone morphogenetic protein-7.

Treatment with BMP-7 causes a shift in the differentiation pathway from myoblastic to osteoblastic in C2C12 mouse myoblast precursor cells in vitro. The underlying molecular mechanism is largely unknown. BMP-7 at 200 ng/ml completely inhibited myotube formation in C2C12 cells and dramatically induced alkaline phosphatase activity up to 20-fold when compared to untreated cells by day 12 in culture. The level of Runx2/Cbfa1 mRNA, a bone-specific transcription factor, was also stimulated up to 6-fold by BMP-7 with a peak at 24 h. In addition BMP-7 treatment stimulated a 55-fold increase in osteocalcin mRNA as early as 24 h after treatment. A novel finding was that the expression of the chondrocyte markers Sox9 and type II collagen was increased as well. Runx2/Cbfa1 is a molecular switch for osteoblast differentiation. To initiate the study of modulators of Runx2/Cbfa1, such as kinases and cofactors, during osteoblastic differentiation of C2C12 cells treated by BMP-7 in vitro, microarray analyses of gene expressions were performed. Microarray data suggested that a total of 882 transcripts were either up- or downregulated at least 2-fold. Cluster analyses revealed 76 genes (including ESTs) with expression patterns that paralleled Runx2/Cbfa1. Thirteen of these 76 genes were initially selected as potential transcription modulators for further study; including CCAAT/enhancer binding protein delta, distal- less homeobox 1, forkhead box F2, insulin-like growth factor binding protein 4, an ortholog of human osteoclast stimulating factor 1 and p300/CBP-associated factor. Some transcription modulators have been associated with osteoblastic differentiation or interacted with Runx2/Cbfa1. Most of them have not been extensively studied in osteoblastic differentiation and in relationship to Runx2/Cbfa1. Thus, these studies identify potential regulators for Runx2/Cbfa1 and osteoblast differentiation. In addition, our data revealed for the first time that BMP-7 not only induced the expression of osteoblastic differentiation markers but also stimulated the expression of chondroblastic markers in C2C12 cells.