Disrupted type II collagenolysis impairs angiogenesis, delays endochondral ossification and initiates aberrant ossification in mouse limbs.

Cartilage remodelling and chondrocyte differentiation are tightly linked to angiogenesis during bone development and endochondral ossification. To investigate whether collagenase-mediated cleavage of the major cartilage collagen (collagen II) plays a role in this process, we generated a knockin mouse in which the mandatory collagenase cleavage site at PQG775↓776LAG, was mutated to PPG775↓776MPG (Col2a1Bailey). This approach blocked collagen II cleavage, and the production of putative collagen II matrikines derived from this site, without modifying matrix metalloproteinase expression or activity. We report here that this mouse (Bailey) is viable. It has a significantly expanded growth plate and exhibits delayed and abnormal angiogenic invasion into the growth plate. Deeper electron microscopy analyses revealed that, at around five weeks of age, a small number of blood vessel(s) penetrate into the growth plate, leading to its abrupt shrinking and the formation of a bony bridge. Our results from in vitro and ex vivo studies suggest that collagen II matrikines stimulate the normal branching of endothelial cells and promote blood vessel invasion at the chondro-osseous junction. The results further suggest that failed collagenolysis in Bailey leads to expansion of the hypertrophic zone and formation of a unique post-hypertrophic zone populated with chondrocytes that re-enter the cell cycle and proliferate. The biological rescue of this in vivo phenotype features the loss of a substantial portion of the growth plate through aberrant ossification, and narrowing of the remaining portion that leads to limb deformation. Together, these data suggest that collagen II matrikines stimulate angiogenesis in skeletal growth and development, revealing novel strategies for stimulating angiogenesis in other contexts such as fracture healing and surgical applications.

Preventive Effects of Poloxamer 188 on Muscle Cell Damage Mechanics Under Oxidative Stress.

High oxidative stress can occur during ischemic reperfusion and chronic inflammation. It has been hypothesized that such oxidative challenges could contribute to clinical risks such as deep tissue pressure ulcers. Skeletal muscles can be challenged by inflammation-induced or reperfusion-induced oxidative stress. Oxidative stress reportedly can lower the compressive damage threshold of skeletal muscles cells, causing actin filament depolymerization, and reduce membrane sealing ability. Skeletal muscles thus become easier to be damaged by mechanical loading under prolonged oxidative exposure. In this study, we investigated the preventive effect of poloxamer 188 (P188) on skeletal muscle cells against extrinsic oxidative challenges (H2O2). It was found that with 1 mM P188 pre-treatment for 1 h, skeletal muscle cells could maintain their compressive damage threshold. The actin polymerization dynamics largely remained stable in term of the expression of cofilin, thymosin beta 4 and profilin. Laser photoporation demonstrated that membrane sealing ability was preserved even as the cells were challenged by H2O2. These findings suggest that P188 pre-treatment can help skeletal muscle cells retain their normal mechanical integrity in oxidative environments, adding a potential clinical use of P188 against the combined challenge of mechanical-oxidative stresses. Such effect may help to prevent deep tissue ulcer development.

Bisphosphonates as a risk factor for adverse orthodontic outcomes: a retrospective cohort study.

INTRODUCTION: Bisphosphonates are a class of drugs commonly prescribed to treat osteoporosis. They act by decreasing the resorption of bone. Since tooth movement depends on bone remodeling, these drugs can impact orthodontic treatment. The purpose of this study was to evaluate the extent to which bisphosphonate therapy is a risk factor for poor orthodontic outcomes. METHODS: Orthodontists were invited to participate in the study by performing case reviews of women over age 50 who were treated from 2002 through 2008. Women who used bisphosphonates were compared with women who did not have a history of bisphosphonate use. Outcomes assessed included treatment time, osteonecrosis of the jaws, incisor alignment, incomplete space closure, and root parallelism. RESULTS: The records for 20 subjects with bisphosphonate exposure were collected, as well as records for 93 subjects without bisphosphonate exposure. In patients undergoing extractions, treatment times were significantly longer if they had a history of bisphosphonate use. No occurrences of osteonecrosis of the jaws were reported, nor did patients end treatment with incisor alignment discrepancies greater than 1 mm, regardless of bisphosphonate exposure. Among patients with extractions or initial spacing, there were higher odds of incomplete space closure (odds ratio, 13) and poor root parallelism (odds ratio, 26) at the end of treatment for patients using bisphosphonates. CONCLUSIONS: Bisphosphonate use is associated with longer treatment times among extraction patients, increased odds of poor space closure, and increased odds of poor root parallelism.

ADAMTS-5: the story so far.

The recent discovery of ADAMTS-5 as the major aggrecanase in mouse cartilage came as a surprise. A great deal of research had focused on ADAMTS-4 and much less was known about the regulation, expression and activity of ADAMTS-5. Two years on, it is still not clear whether ADAMTS-4 or ADAMTS-5 is the major aggrecanase in human cartilage. On the one hand there are in vitro studies using siRNA, neutralising antibodies and immunoprecipitation with anti-ADAMTS antibodies that suggest a significant role for ADAMTS-4 in aggrecanolysis. On the other hand, ADAMTS-5 (but not ADAMTS-4)-deficient mice are protected from cartilage erosion in models of experimental arthritis, and recombinant human ADAMTS-5 is substantially more active than ADAMTS-4. The activity of both enzymes is modulated by C-terminal processing, which occurs naturally in vivo. The most interesting finding to emerge from our comparison of ADAMTS-5 and ADAMTS-4 is that in terms of gene regulation, these two enzymes are the antitheses of each other. In most cases, ADAMTS-5 is constitutively expressed in human chondrocytes and synovial fibroblasts, whereas ADAMTS-4 expression is induced by proinflammatory cytokines. This paper reviews the data on ADAMTS-5 so far. It represents a snapshot in time of a field that is fast-moving and very exciting.

Enantioselective hydrogenation of alpha-aminomethylacrylates containing a free NH group for the synthesis of beta-amino acid derivatives.

We describe highly enantioselective synthesis of beta-amino acid derivatives (1a-c) using asymmetric hydrogenation of alpha-aminomethylacrylates (2a-c), which contain a free basic N H group, as the key step. The alpha-aminomethylacrylates (2a-c) were prepared using the Baylis-Hillman reaction of an appropriate aldehyde with methyl acrylate followed by acetylation of the resulting allylic alcohols (4a-b) and S(N)2'-type amination of the allylic acetates (3a-b).

Oncogenic properties of a novel gene JK-1 located in chromosome 5p and its overexpression in human esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) shows high frequency and mortality in Asian regions, including China. Previous analysis of genomic DNA of ESCC using comparative genomic hybridization indicated that amplification of the chromosome 5p regions is a common event in ESCC cell lines and patient cases of Hong Kong Chinese origin, and the results suggested that the genes located in the chromosome 5p regions may play crucial roles in the molecular pathogenesis of ESCC. Our previous studies on ESCC confirmed the tumorigenic and overexpression properties of a novel gene JS-1 located in chromosome 5p15.2 upstream to delta-catenin. In the present study, another novel gene JK-1 which is located at 5p15.1 downstream to delta-catenin was characterized for its roles in the pathogenesis of ESCC. Thirteen ESCC cell lines and 30 surgical specimens of esophageal tumors were studied for the overexpression of JK-1 using multiplex RT-PCR analysis. The transforming capacity of overexpression of JK-1 was also investigated by transfecting NIH 3T3 and HEK 293 cells with the expression vector cloned with JK-1, followed by the soft agar and foci formation assays. JK-1 was overexpressed in 9/13 (69%) of the ESCC cell lines and 9/30 (30%) of the ESCC patient cases. Both NIH 3T3 and HEK 293 cells acquired the properties of anchorage-dependent and -independent growth when JK-1 was overexpressed. Most significantly, subcutaneous sarcomas were formed in all (3/3) the athymic nude mice after NIH 3T3 cells overexpressing JK-1 were injected subcutaneously. Our results thus indicated that JK-1 is commonly overexpressed in ESCC and has a prominent capacity to transform normal cells. Our overall results thus provide the first evidence that the overexpression of JK-1 and its transforming capacity in normal cells may play a critical role in the molecular pathogenesis of ESCC.

Inhibitory effects of Gleditsia sinensis fruit extract on telomerase activity and oncogenic expression in human esophageal squamous cell carcinoma.

Previous studies have shown that the anomalous fruit extract of Gleditsia sinensis (GSE) exhibited apoptotic properties in various solid and non-solid tumors in vitro. However, the inhibitory actions of GSE on oncogenic expression and telomerase activity in esophageal squamous cell carcinoma (ESCC) have not been studied before. In the present study, the anti-cancer effects of GSE were demonstrated in three ESCC cell lines (HKESC-1, HKESC-2 and SLMT-1) by MTS and anchorage-independent clongen-icity assays, expression studies on oncogenes at 11q13 (CCND1, INT2, FGF4 and EMS1) and real-time quantitative telomeric repeat amplification protocol assay to show the inhibitory effect of GSE on telomerase in ESCC. The means of MTS50 of GSE for the ESCC cell lines and non-tumor NIH 3T3 cells were 21 and 163 microg/ml respectively. The anchorage-independent clongenicity assay showed that SLMT-1 cells lost their colony-forming potential which was dose-dependent to GSE. Moreover, GSE demonstrated dose-dependent suppression on the expression of INT2, EMS1 and FGF4, and inhibition of telomerase activity in the ESCC cell lines. Our overall results thus provide the first evidence that the anti-cancer effects of GSE on ESCC involve the suppression of oncogenic expression and inhibition of telomerase activity. Our findings also offer a new opportunity for the future development of GSE as a novel anti-cancer agent for ESCC and possibly for other cancers.

Position of the American Dietetic Association: the impact of fluoride on health.

The American Dietetic Association reaffirms that fluoride is an important element for all mineralized tissues in the body. Appropriate fluoride exposure and usage is beneficial to bone and tooth integrity and, as such, has an important, positive impact on oral health as well as general health throughout life. Fluoride is an important element in the mineralization of bone and teeth. The proper use of topical and systemic fluoride has resulted in major reductions in dental caries (tooth decay) and its associated disability. The Centers for Disease Control and Prevention have named fluoridation of water as one of the 10 most important public health measures of the 20th century. Nearly 100 national and international organizations recognize the public health benefits of community water fluoridation for preventing dental caries. However, by the year 2000, over one third of the US population (over 100 million people) were still without this critical public health measure. Fluoride also plays a role in bone health. However, the use of high doses of fluoride for prevention of osteoporosis is considered experimental at this point. Dietetics professionals should routinely monitor and promote the use of systemic and topical fluorides, especially in children and adolescents. The American Dietetic Association strongly reaffirms its endorsement of the appropriate use of systemic and topical fluorides, including water fluoridation, at appropriate levels as an important public health measure throughout the life span.

Facile, efficient, and enantiospecific syntheses of 1,1'-N-linked pseudodisaccharides as a new class of glycosidase inhibitors.

This article describes an efficient synthesis of a potent trehalase inhibitor, 1,1'-N-linked pseudodisaccharide 1 (consisting of two valienamines), in 14 steps with an overall yield of 12% and a first synthesis of 2 (consisting of two 2-epi-valienamines) in 15 steps with an overall yield of 24% from (-)-quinic acid. The synthesis involves a stereospecific palladium-catalyzed coupling reaction between an allylic amine and an allylic chloride as the crucial step. The acetonide blocking groups were shown to be the best hydroxyl protecting groups, compatible with the palladium-catalyzed allylic amination reaction that afforded high yields of the 1,1'-N-linked pseudodisaccharides with a minimum amount of an elimination diene side product.

OPENWIDE: an innovative oral health program for non-dental health and human services providers.

Emerging awareness of the nature and severity of oral diseases and disorders and their serious impact on overall health and well-being, combined with a nationwide crisis in access to oral health care for populations with the most and worst disease, makes it imperative that non-dental health and childcare professionals engage more fully in oral health promotion and disease prevention. OPENWIDE is a comprehensive training program designed to help achieve this goal. The Connecticut Department of Public Health has trained more than 2,000 individuals during the first year of the OPENWIDE program. This article reports on successes and impediments to training and implementation encountered in the early stages of OPENWIDE and makes recommendations to improve the curriculum and its delivery to families and children.

Neoepitopes as biomarkers of cartilage catabolism.

Progressive degradation of articular cartilage is a central feature of arthritis and a major determinant of long term joint dysfunction. There are no treatments able to halt the progression of cartilage destruction presently available, and monitoring the benefit of potential therapies is hampered by our inability to measure the "health" of articular cartilage. Serial radiographic assessment of joint space narrowing, the current gold standard, requires measurements over a prolonged time (1-5 years) and is prone to technical difficulties. Other strategies for evaluating cartilage degradation are needed to enable both short and long term monitoring of disease progression and response to therapy. One avenue that holds promise is the use of biomarkers that accurately reflect the degradative state of the articular cartilage. Antibodies that recognise terminal amino acid sequences generated by proteolysis at specific sites in the core protein of both aggrecan and type II collagen (neoepitope antibodies) have become available in recent years. These antibodies have been invaluable for identifying the proteinases responsible for cartilage breakdown both in vitro and in vivo. The presence of neoepitope sequences generated by specific metalloenzyme cleavage of aggrecan and type II collagen correlates well with the progression of cartilage degeneration, both in vitro and in mouse models of arthritis. Preliminary results with quantitative assays of type II collagen neoepitopes suggest that they may be useful markers of joint disease in humans. Long term studies correlating neoepitope concentration with clinical and radiographic disease are now required to validate the utility of neoepitopes as surrogate markers of cartilage degeneration and joint disease.

Generation and novel distribution of matrix metalloproteinase-derived aggrecan fragments in porcine cartilage explants.

We have studied aggrecan catabolism mediated by matrix metalloproteinases (MMPs) in a porcine cartilage culture system. Using antibodies specific for DIPEN(341) and (342)FFGVG neoepitopes, we have detected MMP-derived fragments in conditioned medium and cultured cartilage, by radioimmunoassay, Western blotting, and immunolocalization. Radioimmunoassay revealed that the amount (pmol of epitope/mg of total glycosaminoglycan) of (342)FFGVG epitope released from cartilage remained constant over a 5-day culture period and was not increased by IL-1alpha or retinoate. However, the proportion (pmol of epitope/mg of released glycosaminoglycan) of (342)FFGVG epitope released was decreased upon stimulation, consistent with the involvement of a non-MMP proteinase, such as aggrecanase. The data suggest that in vitro MMPs may be involved in the base-line catabolism of aggrecan. Immunolocalization experiments showed that DIPEN(341) and ITEGE(373) epitopes were increased by treatment with IL-1alpha and retinoate. Confocal microscopy revealed that ITEGE(373) epitope was largely intracellular but with matrix staining in the superficial zone, whereas DIPEN(341) epitope was cell-associated and widely distributed in the matrix. Surprisingly, the majority of (342)FFGVG epitope, determined by radioimmunoassay and Western blotting, was retained in the tissue despite the absence of a G1 domain anchor. Interleukin-1alpha stimulation caused a marked increase in tissue DIPEN(341) and (342)FFGVG epitope, and the (342)FFGVG fragments retained in the tissue were larger than those released into the medium. Active porcine aggrecanase was unable to cleave (342)FFGVG fragments at the downward arrowGlu(373) downward arrowAla(374) bond but cleaved intact aggrecan at this site, suggesting that (342)FFGVG fragments are not substrates for aggrecanase. The apparent retention of large (342)FFGVG fragments within cartilage, and their resistance to N-terminal cleavage by aggrecanase suggests that (342)FF6V6 fragments may have a role in cartilage homeostasis.

Localisation of matrix metalloproteinases and TIMP-2 in resorbing mouse bone.

There is strong evidence that matrix metalloproteinases (MMPs) play a crucial role during osteogenesis and bone remodelling. Their synthesis by osteoblasts has been demonstrated during osteoid degradation prior to resorption of mineralised matrix by osteoclasts and their activities are regulated by tissue inhibitors of metalloproteinases (TIMPs). For this study we developed and utilised specific polyclonal antibodies to assess the presence of collagenase (MMP13), stromelysin 1 (MMP3), gelatinase A (MMP2), gelatinase B (MMP9) and TIMP-2 in both freshly isolated neonatal mouse calvariae and tissues cultured with and without bone-resorbing agents. Monensin was added towards the end of the culture period in order to promote intracellular accumulation of proteins and facilitate antigen detection. In addition, bone sections were stained for the osteoclast marker, tartrate-resistant acid phosphatase (TRAP). In uncultured tissues the bone surfaces had isolated foci of collagenase staining, and cartilage matrix stained for gelatinase B (MMP9) and TIMP-2. Calvariae cultured for as little as 3 h with monensin revealed intracellular staining for MMPs and TIMP-2 in mesenchymal tissues, as well as in cells lining the bone plates. The addition of cytokines to stimulate bone resorption resulted in pronounced TRAP activity along bone surfaces, indicating active resorption. There was a marked upregulation of enzyme synthesis, with matrix staining for collagenase and gelatinase B observed in regions of eroded bone. Increased staining for TIMP-2 was also observed in association with increased synthesis of MMPs. The new antibodies to murine MMPs should prove valuable in future studies of matrix degradation.

Evaluation of some newer matrix metalloproteinases.

Recombinant protein expression techniques have been utilized to facilitate the biochemical and cell biological characterization of human matrix metalloproteinases (MMPs). The importance of the membrane type 1 MMP (MMP 14) in the regulation of pericellular proteolysis, either directly or through the activation of MMP-2, MMP-9, and MMP-13 has been identified. Studies on an in vitro chondrocyte-like cell and an in vivo cartilage repair model indicated that such MT1 MMP-regulated activation cascades are physiologically feasible. MMP19 shows a limited sequence identity with other MMPs and may represent a novel subclass. However, analysis of the recombinant protein identified a number of biochemical properties typical of the MMP family.

Mechanisms for pro matrix metalloproteinase activation.

The activation of pro matrix metalloproteinases (MMPs) by sequential proteolysis of the propeptide blocking the active site cleft is regarded as one of the key levels of regulation of these proteinases. Potential physiological mechanisms including cell-associated plasmin generation by urokinase-like plasminogen activator, or the action of cell surface MT1-MMPs appear to be involved in the initiation of cascades of pro MMP activation. Gelatinase A, collagenase 3 and gelatinase B may be activated by MT-MMP based mechanisms, as evidenced by both biochemical and cell based studies. Hence the regulation of MT-MMPs themselves becomes critical to the determination of MMP activity. This includes activation, assembly at the cell surfaces as TIMP-2 complexes and subsequent inactivation by proteolysis or TIMP inhibition.

Enantiospecific Syntheses of Valienamine and 2-epi-Valienamine(1).

Cyclic sulfite 10, readily available from (-)-quinic acid (3) in 10 steps, was ring opened regio- and stereospecifically with azide anion to give (1S,2R,3R,4R)-1-azido-3,4-di-O-benzyl-5-(benzyloxymethyl)cyclohex-5-ene-2,3,4-triol (11). Deprotection of 11 afforded, for the first time, 2-epi-valienamine (2), which was isolated as penta-N,O-acetyl-2-epi-valienamine (14). The configuration of the free hydroxy group in 11 was inverted by a two-step sequence to give the blocked valienamine 19 that was deprotected to give valienamine (1), isolated as penta-N,O-acetylvalienamine (21). This approach furnished (+)-valienamine (1) in 16 steps (7% overall yield) and recorded the first synthesis of 2-epi-valienamine (2) in 13 steps (11% overall yield).

The activation of ProMMP-2 (gelatinase A) by HT1080 fibrosarcoma cells is promoted by culture on a fibronectin substrate and is concomitant with an increase in processing of MT1-MMP (MMP-14) to a 45 kDa form.

We have assessed the effect of fibronectin and laminin-1 on the expression of molecules involved in the activation pathway of MMP-2, a key proteinase in tissue remodelling. HT1080 fibrosarcoma cells cultured on fibronectin were shown to activate endogenous MMP-2, to a level comparable with that elicited by treatment with phorbol ester. In contrast, the MMP-2 expressed by HT1080 cells cultured on laminin-1 was mainly in the pro- (inactive form). Culture of the cells on peptide fragments of fibronectin derived from the central cell binding domain also promoted MMP-2 activation, indicating that signals via fibronectin binding to integrin receptors may be involved. HT1080 cells cultured on immobilised antibodies to the alpha5 and beta1 integrin subunits secreted levels of active MMP-2 similar to those observed for full length fibronectin, whereas cells cultured on an antibody to the alpha6 integrin subunit secreted mainly proMMP-2. The data demonstrate that the activation of MMP-2 by HT1080 cells is regulated by the nature of the extracellular matrix, and that signals via the alpha5beta1 integrin receptor may be involved in the fibronectin induced up-regulation of MMP-2 activation. We then assessed the effect of fibronectin on the components of the putative MT1-MMP/TIMP-2 'receptor' complex implicated in MMP-2 activation. Levels of TIMP-2 protein expressed by HT1080 cells did not vary detectably between cells cultured on fibronectin or laminin-1. However, the expression of MT1-MMP protein was up-regulated when the cells were cultured on fibronectin, which could be attributed to an increase in levels of a truncated 45 kDa form. Parallel studies using gelatin zymography demonstrated that the up-regulation of the production of the 45 kDa band was concomitant with MMP-2 activation. Inhibitor studies revealed that the truncation of MT1-MMP to a 45 kDa form is MMP mediated, although not inhibited by TIMP-1. In vitro, the 45 kDa form could be generated by cleavage of membrane-bound native MT1-MMP with several recombinant MMPs, including both active MT1-MMP and MMP-2. The implication that either MMP-2 or MT1-MMP can process MT1-MMP to 45 kDa, raises the possibility that truncation of MT1-MMP represents a self-regulatory end-point in the activation pathway of MMP-2.

Induction of matrix metalloproteinase activation cascades based on membrane-type 1 matrix metalloproteinase: associated activation of gelatinase A, gelatinase B and collagenase 3.

SW1353 chondrosarcoma cells cultured in the presence of interleukin-1, concanavalin A or PMA secreted procollagenase 3 (matrix metalloproteinase-13). The enzyme was detected in the culture medium by Western blotting using a specific polyclonal antibody raised against recombinant human procollagenase 3. Oncostatin M enhanced the interleukin-1-induced production of procollagenase 3, whereas interleukin-4 decreased procollagenase 3 synthesis. The enzyme was latent except when the cells had been treated with concanavalin A, when a processed form of 48 kDa, which corresponds to the active form, was found in the culture medium and collagenolytic activity was detected by degradation of 14C-labelled type I collagen. The concanavalin A-induced activation of procollagenase 3 coincided with the processing of progelatinase A (matrix metalloproteinase-2) by the cells, as measured by gelatin zymography. In addition, progelatinase B (matrix metalloproteinase-9) was activated when gelatinase A and collagenase 3 were in their active forms. Concanavalin A treatment of SW1353 cells increased the amount of membrane-type-1 matrix metalloproteinase protein in the cell membranes, suggesting that this membrane-bound enzyme participates in an activation cascade involving collagenase 3 and the gelatinases. This cascade was effectively inhibited by tissue inhibitors of metalloproteinases-2 and -3. Tissue inhibitor of metalloproteinases-1, which is a much weaker inhibitor of membrane-type 1 matrix metalloproteinase than tissue inhibitors of metalloproteinases-2 and -3 [Will, Atkinson, Butler, Smith and Murphy (1996) J. Biol. Chem. 271, 17119-17123], was a weaker inhibitor of the activation cascade.

MT1-MMP on the cell surface causes focal degradation of gelatin films.

The membrane-type matrix metalloproteinases (MT-MMPs) are a subclass of the matrix metalloproteinase (MMP) family which uniquely possess a C-terminal transmembrane domain and are initiators of an activation cascade for progelatinase A (MMP-2). Recent studies have shown that they can also efficiently directly degrade a number of matrix macromolecules. We now show that cells expressing MT1-MMP on their cell surfaces cause subjacent proteolysis of a gelatin film and that this proteolysis is inhibited by TIMP-2 but not by TIMP-1. These data indicate that expression of MT1-MMP on the cell surface may lead to both progelatinase A activation and extracellular matrix degradation.