Mark P. Lewis

Matrix metalloproteinases and oral cancer

For tumours to invade and metastasise, neoplastic cells must be capable of degrading the extracellular matrix (ECM), and accessing blood vessels and lymphatics. This process is mediated in the pericellular environment and is a highly controlled cascade of events utilising the same mechanisms that normal cells use for migrating through tissue barriers, for example, in development and wound healing. Proteolytic enzymes from several families, including matrix metalloproteinases (MMPs), are involved in ECM remodelling. Increased production of these enzymes has been associated with the invasive and/or metastatic phenotype in many tumours. Several MMPs have been shown to play a role in the invasion and metastasis of oral carcinoma, and it is increasingly apparent that tumour cells, as well as producing endogenous MMP, are capable of utilising MMP produced by tumour stromal cells, indicating an active role for stroma in tumour invasion. It is not clear whether a particular invasive system is favoured by oral carcinoma, but it is likely that further understanding of the interactions between carcinoma and stromal cells will provide an opportunity to refine the therapeutic interventions that are currently being tested.

αvβ6integrin promotes invasion of squamous carcinoma cells through up-regulation of matrix metalloproteinase-9

The integrin αvβ6 is a fibronectin receptor, which is not detectable on normal epithelium but is neo‐expressed in oral epithelial dysplasia and oral squamous‐cell carcinoma (SCC), suggesting a role in promoting malignant behaviour and tumour progression. We used transfection and retroviral infection to create a panel of SCC cell lines expressing various levels of αvβ6 to examine this possibility. We found that increased expression of αvβ6 in malignant keratinocytes up‐regulates MMP‐9 and MMP‐2 expression and promotes invasion in an MMP‐9‐dependent manner. Our results suggest a possible mechanism for the involvement of αvβ6 in squamous carcinoma in vivo.

Human adult craniofacial muscle‐derived cells: neural‐cell adhesion‐molecule (NCAM; CD56)‐expressing cells appear to contain multipotential stem cells

Skeletal muscle has been well characterized as a reservoir of myogenic precursors or satellite cells with the potential to participate in cellular repopulation therapies for muscle dysfunction. Recent evidence, however, suggests that the postnatal muscle compartment can be considered an alternative to bone marrow as a source of multipotent cells or muscle‐derived stem cells (MDSCs). MDSCs, when primed with appropriate environmental cues, can differentiate into a variety of non‐muscle cells. The present study describes the application of a new technique for the isolation of adult human myoblasts and putative MDSCs, based on microbead–immunomagnetic selection of CD56+ cells, derived from craniofacial skeletal muscle, and details changes in morphological/molecular phenotype of the purified cells when maintained in either a myogenic or a non‐myogenic milieu. Multiple immunofluorescence microscopy and two‐colour flow‐cytometric analysis of proliferating CD56+ cultures revealed positive staining for myogenic markers (CD56, desmin and M‐cadherin) as well as putative stem‐cell markers [the antigens CD34, CD90 and CD106, and Flk‐1 (fetal liver kinase‐1)/VEGFR‐2 (vascular‐endothelial‐growth‐factor receptor)]. Confluent cultures subjected to cycles of adipogenic or osteogenic induction contained either adipocytes or osteoblasts and myotubes. In conclusion, the CD56+ subpopulation within adult human skeletal muscle is heterogeneous and is composed of both lineage‐committed myogenic cells and multipotent cells (the candidate MDSCs), which are able to form non‐muscle tissue such as fat and bone.

Gelatinase-B (matrix metalloproteinase-9; MMP-9) secretion is involved in the migratory phase of human and murine muscle cell cultures

The remodelling of connective tissue components is a fundamental requirement for a number of pivotal processes in cell biology. These may include myoblast migration and fusion during development and regeneration. In other systems, similar biological processes are facilitated by secretion of the matrix metalloproteinases (MMPs), especially the gelatinases. This study investigated the activity of the gelatinases MMP-2 and 9 by zymography on cell conditioned media in cultures of cells derived from explants of the human masseter muscle and in the murine myoblast cell-line C2C12. Expression of MMP-9 by western blotting and TIMP-1, the major inhibitor of MMPs, by northern blotting, during all phases of myoblast proliferation, migration, alignment and fusion, was also measured. Irrespective of the origin of the cultures, MMP-9 activity was secreted only by single cell and pre-fusion cultures whilst MMP-2 activity was secreted at all stages as well as by myotubes. The loss of MMP-9 activity was due to the loss of MMP-9 protein expression. TIMP-1 mRNA was not detectable at the single cell stage but its expression increased as cells progressed through the pre-fusion and post-fusion stages to reach a maximal in myotube containing cultures. Migration of cells derived from human masseter muscle was inhibited, using a specific anti-MMP-9 blocking monoclonal antibody (6-6B). These data are consistent with the concept that regulation of matrix turnover via MMP-9 may be involved in the events leading to myotube formation, including migration. Loss of expression of this enzyme and expression of TIMP-1 mRNA is associated with myotube containing cultures. Consequently, the ratio between MMPs and TIMPs maybe important in determining myoblast migration and differentiation.

The IGF-I splice variant MGF increases progenitor cells in ALS, dystrophic, and normal muscle

The effects of muscle splice variants of insulin‐like growth factor I (IGF‐I) on proliferation and differentiation were studied in human primary muscle cell cultures from healthy subjects as well as from muscular dystrophy and ALS patients. Although the initial numbers of mononucleated progenitor cells expressing desmin were lower in diseased muscle, the E domain peptide of IGF‐IEc (MGF) significantly increased the numbers of progenitor cells in healthy and diseased muscle. IGF‐I significantly enhances myogenic differentiation whereas MGF E peptide blocks this pathway, resulting in an increased progenitor (stem) cell pool and thus potentially facilitating repair and maintenance of this postmitotic tissue.

αv integrins play an important role in myofibroblast differentiation

Transforming growth factor‐β1 is a potent mediator of the differentiation of fibroblasts into myofibroblasts, which is characterized by the appearance of the cytoskeletal protein α‐smooth muscle actin. The aim of this study was to investigate the role of integrin extracellular matrix receptors in transforming growth factor‐β1–induced myofibroblast differentiation. We show that blockade of the αv and/or β1 integrins prevents the transforming growth factor‐β1–induced myofibroblast differentiation, seen by the increased expression of α‐smooth muscle actin and enhanced collagen gel contraction in three human fibroblast cell lines (from the mouth, skin, and kidney). Further, blockade of αv specific integrins αvβ5 and αvβ3 suppressed myofibroblast differentiation in fibroblasts from the mouth and skin; however, in the kidney cells, the prevention of differentiation was seen only with blockade of αvβ5 integrin but not αvβ3. A possible reason for this result may be different degrees of responsiveness to transforming growth factor‐β1 treatment seen from different anatomical origins of the cell lines. These data indicate a novel role for αv integrins in the differentiation of human fibroblasts from the mouth, skin, and kidney into myofibroblasts and suggest that there is a common differentiation pathway.

Betel-derived alkaloid up-regulates keratinocyte alphavbeta6 integrin expression and promotes oral submucous fibrosis

Oral submucous fibrosis (OSF) is a premalignant, fibrosing disorder of the mouth, pharynx, and oesophagus, with a malignant transformation rate of 7–13%. OSF is strongly associated with areca (betel) nut chewing and worldwide, over 5 million people are affected. As αvβ6 integrin is capable of promoting both tissue fibrosis and carcinoma invasion, we examined its expression in fibroepithelial hyperplasia and OSF. αvβ6 was markedly up‐regulated in OSF, with high expression detected in 22 of 41 cases (p < 0.001). We investigated the functional role of αvβ6 using oral keratinocyte‐derived cells genetically modified to express high αvβ6 (VB6), and also NTERT‐immortalized oral keratinocytes, which express low αvβ6 (OKF6/TERT‐1). VB6 cells showed significant αvβ6‐dependent activation of TGF‐β1, which induced transdifferentiation of oral fibroblasts into myofibroblasts and resulted in up‐regulation of genes associated with tissue fibrosis. These experimental in vitro findings were confirmed using human clinical samples, where we showed that the stroma of OSF contained myofibroblasts and that TGF‐β1‐dependent Smad signalling was detectable both in keratinocytes and in myofibroblasts. We also found that arecoline, the major alkaloid of areca nuts, up‐regulated keratinocyte αvβ6 expression. This was modulated through the M4 muscarinic acetylcholine receptor and was suppressed by the M4 antagonist, tropicamide. Arecoline‐dependent αvβ6 up‐regulation promoted keratinocyte migration and induced invasion, raising the possibility that this mechanism may support malignant transformation. Over 80% of OSF‐related oral cancers examined had moderate/high αvβ6 expression. These data suggest that the pathogenesis of OSF may be epithelial‐driven and involve arecoline‐dependent up‐regulation of αvβ6 integrin. Copyright

Partial characterization of an immortalized human trophoblast cell-line, TCL-1, which possesses a CSF-1 autocrine loop

Many previous studies in both mouse and human placenta have implicated a role for colony stimulating factor-1 (CSF-1) in the regulation of placental development. In this study we have examined CSF-1 production by an immortalized cell line (TCL-1) derived from the choriodecidua, transfected with a retrovirus gene coding for the large-T antigen. TCL-1 cells were uniformly positive by immunocytochemistry for the composite sub-units of human chorionic g gonadotrophin (hCG) but were negative for markers of other cell types localized at the fetal-maternal interface. Gelatinase enzymes were secreted by TCL-1 cells cultured on extracellular matrix in a manner indicative of extra-villous trophoblast. Dot-blot immunoassays and ELISA indicated that CSF-1 was secreted by TCL-1 cells, at levels comparable to primary trophoblast cells and BeWo choriocarcinoma (trophoblast tumour) cells. Reverse transcriptase-polymerase chain reaction analysis confirmed the presence in TCL-1 cells of CSF-1 receptor mRNA (c-fms gene product), indicating that the components of a potential autocrine loop were present in these cells. Proliferation of TCL-1 cells was not affected by the addition of exogenous CSF-1 but was elevated in response to treatment with a CSF-1 neutralizing antibody. The immortalized cell line, TCL-1, provides a potential model in which to investigate regulation of growth and differentiation of trophoblast cells in vitro.

Effect of diabetes and metabolic control on de novo bone formation following guided bone regeneration

OBJECTIVES To evaluate histologically and morphometrically the effect of experimental diabetes and metabolic control on de novo bone formation following guided bone regeneration (GBR). METHODS Thirty-five Wistar rats were allocated in three experimental groups: (a) uncontrolled, streptozotocin-induced diabetes (D); (b) insulin-controlled diabetes (CD); (c) healthy (H). A standardised titanium microimplant with sandblasted and acid-etched surface was placed into the inferior border of the mandible bilaterally. On the test site, the microimplant was covered with a titanium reinforced expanded polytetrafluoroethylene membrane securely fixed in the mandible according to the GBR principle. The contralateral site served as control. Following 90 days of healing, undecalcified sections were prepared and planimetric measurements of the per cent vertical height of newly formed bone and the per cent new bone-to-implant contact were performed. RESULTS In all experimental groups, at the GBR treated sites, significant neo-osteogenesis was observed. The vertical height of the newly formed bone and per cent bone-to-implant contact were not statistically significantly different among the H (51.3 + or - 7.2% and 50 + or - 6.8%), D (30.5 + or - 13.4% and 35 + or - 16.8%) and CD (41.6 + or - 8.3% and 39.9 + or - 6.5%) groups. However, uncontrolled diabetes was related to higher outcome variability and increased rate of infectious complications. In the control sites, marginal bone loss was observed in the D group, whereas, in the H and CD groups, minimal new bone formation was observed. CONCLUSIONS Significant de novo bone formation can be achieved via GBR treatment even in the presence of uncontrolled diabetes, although less predictably compared with the healthy status. Insulin-mediated metabolic control may reverse these adverse effects.

Quantification of Anion and Cation Release from a Range of Ternary Phosphate-based Glasses with Fixed 45 mol% P2O5

This article reports on the use of ion chromatography (IC) to investigate extensively the release profiles of both cations and anions and characterize the relationship between composition and degradation for a ternary-based Na2O-CaO-P2O5 glass system developed as biomaterials. Studies are carried out on glasses with the formula 45P2O5-55(xCaO-Na2O) in deionized water, where x = 30, 35, and 40 mol%, using a cumulative release method, where the solution is changed at regular intervals. Degradation behavior is linear with time where the degradation rate shows an initial decrease with increasing CaO content. This rate then increases with a further addition of CaO. Cation release profiles follow similar trends to the degradation rates. Anion release profiles show a decrease for the PO4 and linear polyphosphate (P2O7 and P3O10) species with increasing CaO content. This decrease is attributed to the cross-linking of the Ca2+ ions. In contrast, the cyclic P3O9 anion exhibits the highest amount of anionic release, which demonstrates similar trends to the cations. These release patterns suggest that the cyclic P3O9 species dominate the degradation rates. The proposed mode of degradation is a hydrolysis reaction, with the cyclic metaphosphate undergoing acid/base catalysis. The pH remains constant for the 30 and 35 mol% CaO glasses, and drops to about 5.5 for the 40 mol% composition. By using a response factor, it is possible to semiquantitatively analyze the additional peaks observed in the chromatograms. Suggestions are also put forward as to the identity of some of these unidentified peaks.