Substance P increases CCN2 dependent on TGF-beta yet Collagen Type I via TGF-beta1 dependent and independent pathways in tenocytes.

Transforming growth factor beta 1 (TGFbeta-1) and connective tissue growth factor (CCN2) are important mediators of tissue repair and fibrosis, with CCN2 functioning as a downstream mediator of TGFß-1. Substance P (SP) is also linked to collagen production in tenocytes. A link between SP, TGFbeta-1 and CCN2 has yet to be established in tenocytes or fibrogenic processes. We sought to determine whether SP induces tenocyte proliferation, CCN2, or collagen production via TGFbeta-1 signaling or independently in rat primary tenocytes. Tenocytes were isolated from rat tendons, cultured and stimulated by SP and/or TGFbeta-1. Cultured cells expressed proteins characteristic of tenocytes (vimentin and tenomodulin) and underwent increased proliferation dose dependently after SP and TGFbeta-1 treatments, alone or combined (more than SP alone when combined). SP induced TGFbeta-1 expression in tenocytes in both dose- and time-dependent manners. SP and TGFbeta-1, alone or combined, stimulated CCN2 expression in tenocytes and their supernatants after both 24 and 48 h of stimulation; a response blocked with addition of a TGFbeta-1 receptor inhibitor. In contrast, SP potentiated collagen type I secretion by tenocytes, a response abrogated by the TGFbeta-1 receptor inhibitor after 48 h of stimulation, but not after the shorter 24 h of stimulation. Our findings suggest that both SP and TGFbeta-1 can stimulate tenocyte fibrogenic processes, albeit differently. TGFbeta-1 pathway signaling was involved in CCN2 production at all time points examined, while SP induced collagen type I production independently prior to the onset of signaling through the TGFbeta-1 pathway.

Increased CCN2, substance P and tissue fibrosis are associated with sensorimotor declines in a rat model of repetitive overuse injury.

Key clinical features of cumulative trauma disorders include pain, muscle weakness, and tissue fibrosis, although the etiology is still under investigation. Here, we characterized the temporal pattern of altered sensorimotor behaviors and inflammatory and fibrogenic processes occurring in forearm muscles and serum of young adult, female rats performing an operant, high repetition high force (HRHF) reaching and grasping task for 6, 12, or 18 weeks. Palmar mechanical sensitivity, cold temperature avoidance and spontaneous behavioral changes increased, while grip strength declined, in 18-week HRHF rats, compared to controls. Flexor digitorum muscles had increased MCP-1 levels after training and increased TNFalpha in 6-week HRHF rats. Serum had increased IL-1beta, IL-10 and IP-10 after training. Yet both muscle and serum inflammation resolved by week 18. In contrast, IFNγ increased at week 18 in both muscle and serum. Given the anti-fibrotic role of IFNγ, and to identify a mechanism for the continued grip strength losses and behavioral sensitivities, we evaluated the fibrogenic proteins CCN2, collagen type I and TGFB1, as well as the nociceptive/fibrogenic peptide substance P. Each increased in and around flexor digitorum muscles and extracellular matrix in the mid-forearm, and in nerves of the forepaw at 18 weeks. CCN2 was also increased in serum at week 18. At a time when inflammation had subsided, increases in fibrogenic proteins correlated with sensorimotor declines. Thus, muscle and nerve fibrosis may be critical components of chronic work-related musculoskeletal disorders. CCN2 and substance P may serve as potential targets for therapeutic intervention, and CCN2 as a serum biomarker of fibrosis progression.

Increased serum and musculotendinous fibrogenic proteins following persistent low-grade inflammation in a rat model of long-term upper extremity overuse.

We examined the relationship between grip strength declines and muscle-tendon responses induced by long-term performance of a high-repetition, low-force (HRLF) reaching task in rats. We hypothesized that grip strength declines would correlate with inflammation, fibrosis and degradation in flexor digitorum muscles and tendons. Grip strength declined after training, and further in weeks 18 and 24, in reach limbs of HRLF rats. Flexor digitorum tissues of reach limbs showed low-grade increases in inflammatory cytokines: IL-1ß after training and in week 18, IL-1α in week 18, TNF-α and IL-6 after training and in week 24, and IL-10 in week 24, with greater increases in tendons than muscles. Similar cytokine increases were detected in serum with HRLF: IL-1α and IL-10 in week 18, and TNF-α and IL-6 in week 24. Grip strength correlated inversely with IL-6 in muscles, tendons and serum, and TNF-α in muscles and serum. Four fibrogenic proteins, TGFB1, CTGF, PDGFab and PDGFbb, and hydroxyproline, a marker of collagen synthesis, increased in serum in HRLF weeks 18 or 24, concomitant with epitendon thickening, increased muscle and tendon TGFB1 and CTGF. A collagenolytic gelatinase, MMP2, increased by week 18 in serum, tendons and muscles of HRLF rats. Grip strength correlated inversely with TGFB1 in muscles, tendons and serum; with CTGF-immunoreactive fibroblasts in tendons; and with MMP2 in tendons and serum. Thus, motor declines correlated with low-grade systemic and musculotendinous inflammation throughout task performance, and increased fibrogenic and degradative proteins with prolonged task performance. Serum TNF-α, IL-6, TGFB1, CTGF and MMP2 may serve as serum biomarkers of work-related musculoskeletal disorders, although further studies in humans are needed.

Pharmacodynamics of recombinant human erythropoietin in murine bone marrow.

PURPOSE: Originally approved for three times/week dosing, recombinant human erythropoietin (rhEPO) is now often used at weekly intervals. We have studied rhEPO in mice to better understand why the extended dosing interval retains efficacy. METHODS: C57Bl/6 mice received a single sc. dose of rhEPO (3,000 IU/kg). Bone marrow and blood were collected at 8 h and 1, 2, 5 and 7 days. Staining for TER-119 and CD71, pulse labeling with bromodeoxyuridine, annexin-V binding and vital staining with 7-aminoactinomycin D: were used cell cycle and apoptosis in erythroblasts by four color flow cytometry. RESULTS: A wave of proliferation and/or maturation progressed through all erythroblasts, resulting in the emigration of immature reticulocytes into the periphery. An increase in the fraction of erythroblasts in S and G2M was found, but suppression of apoptosis was not. CONCLUSIONS: Most of the effects of rhEPO occurred 48 h after dosing, when the concentration of rhEPO was less than 1% of Cmax, suggesting that the processes set in motion by rhEPO can continue after rhEPO concentrations fall. Our observation of apoptosis in erythroblasts even when rhEPO concentrations were high suggests that regulatory mechanisms which down-regulate erythropoiesis are also engaged.

Highly potent inhibitors of methionine aminopeptidase-2 based on a 1,2,4-triazole pharmacophore.

High-throughput screening for inhibitors of the human metalloprotease, methionine aminopeptidase-2 (MetAP2), identified a potent class of 3-anilino-5-benzylthio-1,2,4-triazole compounds. Efficient array and interative synthesis of triazoles led to rapid SAR development around the aniline, benzylthio, and triazole moeities. Evaluation of these analogs in a human MetAP2 enzyme assay led to the identification of several inhibitors with potencies in the 50-100 picomolar range. The deleterious effects on inhibitor potency by methylation of the anilino-triazole nitrogens, as well as the X-ray crystal structure of triazole 102 bound in the active site of MetAP2, confirm the key interactions between the triazole nitrogens, the active site cobalt atoms, and the His-231 side-chain. The structure has also provided a rationale for interpreting SAR within the triazole series. Key aniline (2-isopropylphenyl) and sulfur substituents (furanylmethyl) identified in the SAR studies led to the identification of potent inhibitors (103 and 104) of endothelial cell proliferation. Triazoles 103 and 104 also exhibited dose-dependent activity in an aortic ring tissue model of angiogenesis highlighting the potential utility of MetAP2 inhibitors as anticancer agents.

Haematogenous metastasis to ascending colon in a patient with hepatocellular carcinoma and autoimmune hepatitis.

The association of hepatocellular carcinoma with chronic active autoimmune hepatitis and haematogenous metastasis to ascending colon has not been reported previously in the literature. The patient was asymptomatic for colonic disease and the finding of colonic involvement was incidental on scans subsequently confirmed by colonoscopy. Usually hepatocellular tumour mass would involve the colon by direct contiguity. Owing to haematogenous metastasis, which is extremely rare, the colonic mass was discontiguous from hepatic tumour lesions. The unique presentation of this case and the differential diagnosis of hepatic tumours coexisting with colonic tumours are highlighted in this case report.

A statistical pattern recognition approach for determining cellular viability and lineage phenotype in cultured cells and murine bone marrow.

BACKGROUND: Cellular binding of annexin V and membrane permeability to 7-aminoactinomycin D (7AAD) are important tools for studying apoptosis and cell death by flow cytometry. Combining viability markers with cell surface marker expression is routinely used to study various cell lineages. Current classification methods using strict thresholds, or "gates," on the fluorescent intensity of these markers are subjective in nature and may not fully describe the phenotypes of interest. We have developed objective criteria for phenotypic boundary recognition through the application of statistical pattern recognition. This task was achieved using artificial neural networks (ANNs) that were trained to recognize subsets of cells with known phenotypes, and then used to determine decision boundaries based on statistical measures of similarity. This approach was then used to test the hypothesis that erythropoietin (EPO) inhibits apoptosis and cell death in erythroid precursor cells in murine bone marrow. METHODS: Our method was developed for classification of viability using an in vitro cell system and then applied to an ex vivo analysis of murine late-stage erythroid progenitors. To induce apoptosis and cell death in vitro, an EPO-dependent human leukemic cell line, UT-7(EPO) cells were incubated without recombinant human erythropoietin (rhEPO) for 72 h. Five different ANNs were trained to recognize live, apoptotic, and dead cells using a "known" subset of the data for training, and a K-fold cross validation procedure for error estimation. The ANNs developed with the in vitro system were then applied to classify cells from an ex vivo study of rhEPO treated mice. Tg197 (human tumor necrosis-alpha transgenic mice, a model of anemia of chronic disease) received a single s.c. dose of 10,000 U/kg rhEPO and femoral bone marrow was collected 1, 2, 4, and 8 days after dosing. Femoral bone marrow cells were stained with TER-119 PE, CD71 APC enable identification of erythroid precursors, and annexin V FITC and 7AAD to identify the apoptotic and dead cells. During classification forward and side angle light scatter were also input to all pattern recognition systems. RESULTS: Similar decision boundaries between live, apoptotic, and dead cells were consistently identified by the neural networks. The best performing network was a radial basis function multi-perceptron that produced an estimated average error rate of 4.5% +/- 0.9%. Using these boundaries, the following results were reached: depriving UT-7(EPO) cells of rhEPO induced apoptosis and cell death while the addition of rhEPO rescued the cells in a dose-dependent manner. In vivo, treatment with rhEPO resulted in an increase of live erythroid cells in the bone marrow to 119.8% +/- 9.8% of control at the 8 day time point. However, a statistically significant transient increase in TER-119(+) CD71(+) 7AAD(+) dead erythroid precursors was observed at the 1 and 2 day time points with a corresponding decrease in TER-119(+) CD71(+) 7AAD(-) Annexin V(-) live erythroid precursors, and no change in the number of TER-119(+) CD71(+) annexin V(+) 7AAD(-) apoptotic erythroid precursors in the bone marrow. CONCLUSIONS: A statistical pattern recognition approach to viability classification provides an objective rationale for setting decision boundaries between "positive" and "negative" intensity measures in cytometric data. Using this approach we have confirmed that rhEPO inhibits apoptosis and cell death in an EPO dependent cell line in vitro, but failed to do so in vivo, suggesting EPO may not act as a simple antiapoptotic agent in the bone marrow. Rather, homeostatic mechanisms may regulate the pharmacodynamic response to rhEPO.

4-Aryl-1,2,3-triazole: a novel template for a reversible methionine aminopeptidase 2 inhibitor, optimized to inhibit angiogenesis in vivo.

Inhibitors of human methionine aminopeptidase type 2 (hMetAP2) are of interest as potential treatments for cancer. A new class of small molecule reversible inhibitors of hMetAP2 was discovered and optimized, the 4-aryl-1,2,3-triazoles. Compound 24, a potent inhibitor of cobalt-activated hMetAP2, also inhibits human and mouse endothelial cell growth. Using a mouse matrigel model, this reversible hMetAP2 inhibitor was also shown to inhibit angiogenesis in vivo.

A novel site contributing to growth-arrest-specific gene 6 binding to its receptors as revealed by a human monoclonal antibody.

Gas6 (growth-arrest-specific gene 6) is a vitamin K-dependent protein known to activate the Axl family of receptor tyrosine kinases. It is an important regulator of thrombosis and many other biological functions. The C-terminus of Gas6 binds to receptors and consists of two laminin-like globular domains LG1 and LG2. It has been reported that a Ca2+-binding site at the junction of LG1 and LG2 domains and a hydrophobic patch at the LG2 domain are important for receptor binding [Sasaki, Knyazev, Cheburkin, Gohring, Tisi, Ullrich, Timpl and Hohenester (2002) J. Biol. Chem. 277, 44164-44170]. In the present study, we developed a neutralizing human monoclonal antibody, named CNTO300, for Gas6. The antibody was generated by immunization of human IgG-expressing transgenic mice with recombinant human Gas6 protein and the anti-Gas6 IgG sequences were rescued from an unstable hybridoma clone. Binding of Gas6 to its receptors was partially inhibited by the CNTO300 antibody in a dose-dependent manner. To characterize further the interaction between Gas6 and this antibody, the binding kinetics of CNTO300 for recombinant Gas6 were compared with independently expressed LG1 and LG2. The CNTO300 antibody showed comparable binding affinity, yet different dependence on Ca2+, to Gas6 and LG1. No binding to LG2 was detected. In the presence of EDTA, binding of the antibody to Gas6 was disrupted, but no significant effect of EDTA on LG1 binding was evident. Further epitope mapping identified a Gas6 peptide sequence recognized by the CNTO300 antibody. This peptide sequence was found to be located at the LG1 domain distant from the Ca2+-binding site and the hydrophobic patch. Co-interaction of Gas6 with its receptor and CNTO300 antibody was detected by BIAcore analysis, suggesting a second receptor-binding site on the LG1 domain. This hypothesis was further supported by direct binding of Gas6 receptors to an independently expressed LG1 domain. Our results revealed, for the first time, a second binding site for Gas6-receptor interaction.

Chfr regulates a mitotic stress pathway through its RING-finger domain with ubiquitin ligase activity.

Resistance to chemotherapy targeting microtubules could be partially because of the delay in chromosome condensation and segregation during mitosis. The Chfr pathway has been defined recently, and its activation causes a delay in chromosome condensation in response to mitotic stress. Because Chfr contains a RING-finger domain, we tested whether Chfr inhibits chromosome condensation through an ubiquitin (ubiquitin)-dependent pathway. In the presence of purified E1, Ubc4, or Ubc5, and ubiquitin, Chfr catalyzes its own ubiquitination in vitro, an activity requiring the RING domain. In vivo, overexpressed Chfr but not a RING domain mutant is spontaneously ubiquitinated. Our studies with DLD1 cells stably expressing wild-type Chfr and Chfr lacking the RING domain indicated that the RING-finger deletion mutant was defective in inhibiting chromosome condensation after Taxol treatment. In addition, Chfr expression increases the survival rate after Taxol treatment, an activity requiring the RING domain. Preliminary studies indicate that Chfr expression is cell cycle regulated and is dependent on its ubiquitin ligase activity. It is very likely that the Chfr-mediated ubiquitin-dependent pathway is a critical component of the response to mitotic stress.