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Vasculogenic mimicry (VM) is the formation of a blood supply system that confers aggressive and metastatic properties to tumors and correlates with a poor prognosis in cancer patients. Thus, the inhibition of VM is considered an effective approach for cancer treatment, although such a mechanism remains poorly described. In the present study, we examined methionine aminopeptidase‑2 (MetAP2), a key factor of angiogenesis, and demonstrated that it is pivotal for VM, using pharmacological and genetic approaches. Fumagillin and TNP‑470, angiogenesis inhibitors that target MetAP2, significantly suppressed VM in various human cancer cell lines. We established MetAP2‑knockout (KO) human fibrosarcoma HT1080 cells using the CRISPR/Cas9 system and found that VM was attenuated in these cells. Furthermore, re‑expression of wild‑type MetAP2 restored VM in the MetAP2‑KO HT1080 cells, but the substitution of D251, a conserved amino acid in MetAP2, failed to rescue the VM. Collectively, our results demonstrate that MetAP2 is critical for VM in human cancer cells and suggest fumagillin and TNP‑470 as potent VM‑suppressing agents.

Mitochondria evolved from endosymbiotic bacteria to become essential organelles of eukaryotic cells. The unique lipid composition and structure of mitochondrial membranes are critical for the proper functioning of mitochondria. However, stress responses that help maintain the mitochondrial membrane integrity are not well understood. One reason for this lack of insight is the absence of efficient tools to specifically damage mitochondrial membranes. Here, through a compound screen, we found that two bis-biguanide compounds, chlorhexidine and alexidine, modified the activity of the inner mitochondrial membrane (IMM)-resident protease OMA1 by altering the integrity of the IMM. These compounds are well-known bactericides whose mechanism of action has centered on their damage-inducing activity on bacterial membranes. We found alexidine binds to the IMM likely through the electrostatic interaction driven by the membrane potential as well as an affinity for anionic phospholipids. Electron microscopic analysis revealed that alexidine severely perturbated the cristae structure. Notably, alexidine evoked a specific transcriptional/proteostasis signature that was not induced by other typical mitochondrial stressors, highlighting the unique property of alexidine as a novel mitochondrial membrane stressor. Our findings provide a chemical-biological tool that should enable the delineation of mitochondrial stress-signaling pathways required to maintain the mitochondrial membrane homeostasis.

Ultraviolet B radiation represents 10% of the total UV radiation that reaches the Earth's surface, being the primary responsible for the biological effects related to skin cancer and photoaging. Ilex Paraguariensis A. St. Hil., known as Yerba mate (YM), is a native tree of South America whose polyphenols in its leaves are described to exhibit photochemoprotective effect and are employed in the treatment of cancer. Additionally, the polyphenols are used to prevent lipid peroxidation and reduce the UV-induced damage, which ultimately decreases the oxidative stress. Thus, the present study aimed to characterize a new YM extract, evaluate the extract cytotoxicity and develop a formulation containing YM extract to prevent UVB-induced damage in mice skin. The YM extract showed high levels of polyphenols, flavonoids, and tannins and exhibited excellent antioxidant activity. Its main components were suggested as chlorogenic acid (1.92%) and caffeic acid (0.41%). Besides, YM extract did not exhibit cytotoxicity in fibroblasts and decreased the activity of myeloperoxidase and metalloproteinase-2 after acute UVB exposure. As a result, the formulation containing the YM extract showed a potential photochemoprotective., (Copyright © 2018. Published by Elsevier B.V.)

Arteries from young healthy animals respond to chronic changes in blood flow and blood pressure by structural remodeling. We tested whether the ability to respond to decreased (-90%) or increased (+100%) blood flow is impaired during the development of deoxycorticosterone acetate (DOCA)-salt hypertension in rats, a model for an upregulated endothelin-1 system. Mesenteric small arteries (MrA) were exposed to low blood flow (LF) or high blood flow (HF) for 4 or 7 weeks. The bioavailability of vasoactive peptides was modified by chronic treatment of the rats with the dual neutral endopeptidase (NEP)/endothelin-converting enzyme (ECE) inhibitor SOL1. After 3 or 6 weeks of hypertension, the MrA showed hypertrophic arterial remodeling (3 weeks: media cross-sectional area (mCSA): 10±1 × 10(3) to 17±2 × 10(3) μm(2); 6 weeks: 13±2 × 10(3) to 24±3 × 10(3) μm(2)). After 3, but not 6, weeks of hypertension, the arterial diameter was increased (Ø: 385±13 to 463±14 μm). SOL1 reduced hypertrophy after 3 weeks of hypertension (mCSA: 6 × 10(3)±1 × 10(3) μm(2)). The diameter of the HF arteries of normotensive rats increased (Ø: 463±22 μm) but no expansion occurred in the HF arteries of hypertensive rats (Ø: 471±16 μm). MrA from SOL1-treated hypertensive rats did show a significant diameter increase (Ø: 419±13 to 475±16 μm). Arteries exposed to LF showed inward remodeling in normotensive and hypertensive rats (mean Ø between 235 and 290 μm), and infiltration of monocyte/macrophages. SOL1 treatment did not affect the arterial diameter of LF arteries but reduced the infiltration of monocyte/macrophages. We show for the first time that flow-induced remodeling is impaired during the development of DOCA-salt hypertension and that this can be prevented by chronic NEP/ECE inhibition.

Objective: Recanalization therapies for ischemic stroke have been slow to change clinical practice because of perceived and published risks of hemorrhage associated with lytic administration. We quantified alfimeprase in an acute ischemia-reperfusion model, as compared with recombinant tissue plasminogen activator, with hemorrhagic transformation as the primary endpoint and infarction volume and blood-brain barrier permeability as secondary endpoints., Methods: Five groups were studied in a blinded fashion: alfimeprase at doses of 0.03 (n=8), 0.1 (n=11) and 0.3 mg/kg (n=8); recombinant tissue plasminogen activator at 1 mg/kg (n=9); carrier infused controls (n=9). The middle cerebral artery was occluded for 5 hours followed by removal of the suture for reperfusion. Drugs were infused immediately following reperfusion over a 10-minute period. Approximately 24 hours later, the animals were anesthetized and decapitated, and the brains were rapidly harvested and frozen. Serial brain sections were obtained and inspected for hemorrhages. Infarction and blood-brain barrier permeability were also evaluated in additional experiments in control, 0.1 mg/kg alfimeprase and 1 mg/kg recombinant tissue plasminogen activator-treated rats., Results: The hemorrhagic transformation frequency, neurological deficit and the mortality rate of alfimeprase were significantly lower than for recombinant tissue plasminogen activator at the 0.03 mg/kg dose and not statistically different at the higher doses. Infarction and blood-brain barrier permeability were not significantly different among control, 0.1 mg/kg alfimeprase and recombinant tissue plasminogen activator., Discussion: In this model, alfimeprase, a new fibrinolytic agent, exhibits a profile comparable to recombinant tissue plasminogen activator.

Tumor progress depends on the proliferation of cancer cells, their interactions with stroma and the proteolytic action of enzymes. Colon cancer is c-kit positive and responsive to the specific tyrosine kinase inhibitor imatinib. We investigated the effect of imatinib on the proliferation of a panel of epithelial colon cancer cell lines in presence and absence of the antimetabolite 5-FU, and the effect of conditioned media (CM) derived from colon stromal fibroblasts with and without previous exposure to imatinib. The effects of imatinib on gene expression of MMPs and TIMPs were also studied. Imatinib effectively inhibited the proliferation of all cell lines, showing IC(50) from 0.3 to 3 microM. Its combination with 5-FU significantly enhances the growth inhibition of the highly tumourigenic HT-29 cells. CM derived from stromal fibroblasts induced the proliferation of the HT-29 cells; this stimulatory effect was abolished upon treatment with CM obtained after exposure of fibroblasts to imatinib. Gene expression of MT1-, MT2-MMP and MMP-7 was also inhibited depending on the cell line, whereas that of TIMP-2 was not affected. CM stimulated MT1-MMP protein expression by HT-29; this stimulatory effect was suppressed in the presence of imatinib. Activation of pro-MMP2 to MMP2 in culture medium of HT-29 treated with CM was increased and this activity was inhibited in presence of imatinib. The obtained data showed that imatinib is a powerful inhibitor of human colon cancer cell growth and effectively suppresses the stromal-induced stimulation of cancer cell growth and activation of proMMP2. Further studies are warranted to evaluate the in vivo effects., ((c) 2007 Wiley-Liss, Inc.)

Background and Purpose: CGS-26303 inhibits endothelin converting enzyme (ECE)-1 more specifically than phosphoramidon. We have studied the effect of CGS-26303 on ECE-1 expression in bovine aortic endothelial cells., Methods: ECE-1 activity and big endothelin (ET)-1 levels were measured by ELISA, ECE-1 expression using western and northern blot and promoter activity using transfection assays., Key Results: ECE-1 activity was completely inhibited by CGS-26303 25 microM and phosphoramidon 100 microM. CGS-26303 and phosphoramidon, though not thiorphan, a neutral endopeptidase (NEP) inhibitor, stimulated ECE-1 expression in cells (maximal effect at 16 h, 25 microM). Cycloheximide abolished that effect. CGS-26303 induced ECE-1 mRNA expression and ECE-1 promoter activity. CGS-35066, a selective ECE-1 inhibitor, mimicked the effects of CGS-26303, suggesting that the effect was specific to ECE-1 inhibition. Big ET-1 accumulated in the cells and in the supernatants after CGS-26303 treatment. Neither exogenously added ET-1 nor the blockade of their receptors with bosentan modified ECE-1 protein. When big ET-1 was added to cells, significant increases in ECE-1 protein content and ECE-1 promoter activity were found. Bosentan did not block those effects. CGS-26303 did not modify prepro-ET-1 expression. CGS-26303 and big ET-1 induced the same effects in human endothelial cells, at lower doses., Conclusions: These results suggest that the accumulation of big ET-1 is responsible for the effects of CGS-26303 on ECE-1 and they did not depend on NEP blockade. Changes in ECE-1 protein after the administration of CGS-26303 could lead to a decreased response in long-term treatments.

The local and systemic pathophysiological alterations induced by BjussuSP-I, a thrombin-like serine proteinase from the venom of the snake Bothrops jararacussu, were assessed in mice. BjussuSP-I induced a mild edema but no local myonecrosis or hemorrhage. It did not induce any microvascular alteration in the cremaster muscle. Intramuscular injection of BjussuSP-I promoted an increase in the expression of proMMP-9, but it did not induce the activation of proMMP-2 or proMMP-9 synthesized in muscle tissue injected with a myotoxic phospholipase A(2) homolog. BjussuSP-I induced defibrin(ogen)ation upon intravenous and intramuscular injections, with reduction in plasma fibrinogen concentration and increments in the levels of fibrin degradation products and D-dimer. When compared with animals having normal coagulation, mice defibrin(ogen)ated by BjussuSP-I developed a slightly larger hemorrhagic lesion in the skin when injected with metalloproteinase BaP1. Intravenous injection of sublethal doses of BjussuSP-I promoted a series of behavioral and motor changes similar to those previously described for 'gyroxin', i.e. opisthotonus and a circular body movement along the longitudinal axis.

Methionine aminopeptidases (MetAP) are proteases which remove the N-terminal methionine from newly synthesized proteins. Associations of MetAP2 with tumor progression of different cancers have been repeatedly reported. We aim to determine if MetAP2 is expressed in cholangiocarcinomas (CCA) and investigate to see if it would be a useful therapeutic target. We evaluated MetAP2 expression by immunohistochemistry in 82 patients of intrahepatic CCA. MetAP2 was expressed in bile ducts to various degrees. It was occasionally expressed with weak staining in normal bile duct epithelium but was strikingly over-expressed in dysplastic bile duct epithelia, primary and metastatic CCA tissues (p < 0.001). The increased expression of MetAP2 in proliferating bile duct was evident. All metastatic tumors had stronger expression of MetAP2 than the corresponding primary tumors. Fumagillin, a MetAP2 specific inhibitor, significantly inhibited cell proliferation in dose dependent manner and the degree of growth inhibition was dependent on the amount of cellular enzyme. The present study highlights the involvement of MetAP2 in an early event of carcinogenesis of CCA. The findings represent the first description of increased MetAP2 expression in CCA. The inhibition of enzyme activity using MetAP2 inhibitors may be a potential strategy for long-term control of tumor development and progression in CCA patients.

DGR [staphylokinase (Sak) variant K35R, in which lysine (K) at position 35 is replaced by arginine (R)], a recombinant mutant of the Staphylococcus aureus enzyme, is a promising drug for thrombotic disorders. In the present work, DGR was successfully overexpressed by the plasmid JF1125[pST-DGR] as a soluble cytoplasmic protein in a 30-litre fermentor that accounted for more than 50% of the total cellular protein. The expressed DGR was subsequently purified by using a simple three-step chromatographic purification process developed at a pilot scale. The clearance of host-cell-protein contaminants in the protein purification process was confirmed by SDS/PAGE and Western blotting, using rabbit antisera raised against Escherichia coli JF1125 cell proteins. SDS/PAGE, isoelectric focusing and HPLC-MS analysis indicated that the purified DGR is almost completely homogeneous. The purification process resulted in greater-than-98% pure DGR and yielded up to 25.0 mg/g wet weight of cells. The effect of pH and temperature on the stability of DGR was investigated further. The results showed that DGR was highly stable at neutral pH and more stable than two other wild-type Saks, SakSTAR and Sak42D, when submitted to high temperatures.

Alport syndrome is a glomerular basement membrane (GBM) disease caused by mutations in type IV collagen genes. A unique irregular thickening and thinning of the GBM characterizes the progressive glomerular pathology. The metabolic imbalances responsible for these GBM irregularities are not known. Here we show that macrophage metalloelastase (MMP-12) expression is >40-fold induced in glomeruli from Alport mice and is markedly induced in glomeruli of both humans and dogs with Alport syndrome. Treatment of Alport mice with MMI270 (CGS27023A), a broad spectrum MMP inhibitor that blocks MMP-12 activity, results in largely restored GBM ultrastructure and function. Treatment with BAY-129566, a broad spectrum MMP inhibitor that does not inhibit MMP-12, had no effect. We show that inhibition of CC chemokine receptor 2 (CCR2) receptor signaling with propagermanium blocks induction of MMP-12 mRNA and prevents GBM damage. CCR2 receptor is expressed in glomerular podocytes of Alport mice, suggesting MCP-1 activation of CCR2 on podocytes may underlie induction of MMP-12. These data indicate that the irregular GBM that characterizes Alport syndrome may be mediated, in part, by focal degradation of the GBM due to MMP dysregulation, in particular, MMP-12. Thus, MMP-12/CCR2 inhibitors may provide a novel and effective therapeutic stra-tegy for Alport glomerular disease.

The StcE zinc metalloprotease is secreted by enterohemorrhagic Escherichia coli (EHEC) O157:H7 and contributes to intimate adherence of this bacterium to host cells, a process essential for mammalian colonization. StcE has also been shown to localize the inflammatory regulator C1 esterase inhibitor (C1-INH) to cell membranes. We tried to more fully characterize StcE activity to better understand its role in EHEC pathogenesis. StcE was active at pH 6.1 to 9.0, in the presence of NaCl concentrations ranging from 0 to 600 mM, and at 4 degrees C to 55 degrees C. Interestingly, antisera against StcE or C1-INH did not eliminate StcE cleavage of C1-INH. Treatment of StcE with the proteases trypsin, chymotrypsin, human neutrophil elastase, and Pseudomonas aeruginosa elastase did not eliminate StcE activity against C1-INH. After StcE was kept at 23 degrees C for 65 days, it exhibited full proteolytic activity, and it retained 30% of its original activity after incubation for 8 days at 37 degrees C. Together, these results show the StcE protease is a stable enzyme that is probably active in the environment of the colon. Additionally, k(cat)/K(m) data showed that StcE proteolytic activity was 2.5-fold more efficient with the secreted mucin MUC7 than with the complement regulator C1-INH. This evidence supports a model which includes two roles for StcE during infection, in which StcE acts first as a mucinase and then as an anti-inflammatory agent by localizing C1-INH to cell membranes.

The regulatory mechanisms of neuropeptide-metabolizing enzymes often play a critical role in the pathogenesis of neuronal damage. A systemic administration of pentylenetetrazol (PTZ), an antagonist of GABA(A) receptor ion channel binding site, causes generalized epilepsy in an animal model. In the present study, we examined the involvement of prolyl oligopeptidase (POP), thimet oligopeptidase/neurolysin (EP 24.15/16) and glial proteins in PTZ-treated rat brain regions, and the suppressive effect of MK-801, a non-competitive NMDA receptor antagonist, pretreatment for their proteins. The activity of POP significantly decreased in the hippocampus at 30min and 3h, and in the frontal cortex at 3h after PTZ treatment, and pretreatment with MK-801 recovered the activity in the cortex at 3h. The activity of EP 24.15/16 significantly decreased in the hippocampus at 3h and 1 day, and in the cortex at 3h after the PTZ administration, whereas pretreatment with MK-801 recovered the change of the activity. The Western blot analysis of EP 24.15 showed significant decrease of the protein level in the hippocampus 3h after the PTZ treatment, whereas pretreatment with MK-801 recovered. The expression of GFAP and CD11b immunohistochemically increased in the hippocampus of the PTZ-treated rat as compared with controls. Pretreatment with MK-801 also recovered the GFAP and CD11b expression. These data suggest that PTZ-induced seizures of the rats cause indirect activation of glutamate NMDA receptors, then decrease POP and EP 24.15/16 enzyme activities and EP 24.15 immunoreactivity in the neuronal cells of the hippocampal formation. We speculate that changes of those peptidases in the brain may be related to the levels of the neuropeptides regulating PTZ-induced seizures.

Objective: Endothelin-1 (ET-1), a key mediator of inflammatory processes associated with bacterial infection, is a 21-amino acid peptide produced from a biologically inactive big ET-1 by the action of endothelin-converting enzyme-1 (ECE-1) that acts through G protein-coupled ET(A) and ET(B) receptors. Here we report on the role of ET-1 in the mediation of the detrimental influence of Helicobacter pylori on the synthesis of gastric mucin., Material and Methods: Rat gastric mucosal cells were exposed to H. pylori key virulence factor, lipopolysaccharide (LPS)., Results: The LPS inhibitory effect on gastric mucin synthesis was accompanied by a marked increase in ET-1 generation and enhancement in ECE-1 activity. Inhibition of ECE-1 with phosphoramidon not only led to the impedance of LPS-induced ET-1 generation, but also countered the detrimental effect of LPS on mucin synthesis. Moreover, the LPS inhibitory effect on mucin synthesis was blocked by ET(A) receptor antagonist BQ610, but not by ET(B) receptor antagonist BQ788. Furthermore, the LPS-induced suppression in gastric mucin synthesis was countered in a concentration-dependent fashion by PD153035 (81.7%), a specific inhibitor of epidermal growth factor receptor (EGFR) kinase as well as PP2 (69.8%), a selective inhibitor of tyrosine kinase Src responsible for ligand-independent EGFR transactivation., Conclusions: Our findings are the first to show that the detrimental effect of H. pylori on gastric mucin synthesis is intimately linked to the events associated with ECE-1 up-regulation, enhancement in ET-1 production, and G protein-coupled ET(A) receptor activation that triggers the EGFR transactivation.

Although matrilysin (MMP-7) is overexpressed in various malignancies, few studies have evaluated its role in epithelial ovarian cancer (EOC) invasion and metastasis. We report that the secretion of MMP-7 in EOC is stimulated significantly by vascular endothelial growth factor (VEGF) and interlukin-8 (IL-8). We also examined the in vivo expression of MMP-7 in EOC and its effects on the in vitro invasion and progelatinase activation. We report that MMP-7 is overexpressed in ovarian cancer cell lines and EOC surgical specimens. DOV13 cells incubated with active rhMMP-7 significantly increased cellular invasion and proMMP-2 activation. RhMMP-7 also showed the ability to activate proMMP-2 and proMMP-9 in immortalized ovarian epithelial cell (IOSE-29) conditioned medium. In addition, rhMMP-7 was able to activate progelatinase in a concentration-dependent manner in vitro. TIMP-2 or the generic MMP inhibitor-GM6001 inhibited both the activation of proMMP-2 and the increased invasion of DOV13 cells promoted by rhMMP-7. By incubation of MMP2-TIMP-2 complex with equal molar rhMMP-7, MMP-2 was dissociated from the complex and activated in a time-dependent manner, suggesting that TIMP-2 helps to keep the latency of MMP-2. TIMP-2 also showed inhibitory effects on the MMP-7 induced increase of gelatinolytic activity in DOV13 and IOSE-29 conditioned media. A strong co-localization of MMP-7 and MMP-2 was observed in DOV13 cells and ovarian carcinoma permanent tissue sections. These results indicate MMP-7 is overexpressed in malignant ovarian epithelium and suggest MMP-7 may facilitate tumor cell invasion in vivo partly through the induction of progelatinase activation.

Alpha-lipoic acid (LA) is a disulphide-containing fatty acid that is absorbed from the diet and transported to tissues. Once it has been taken up by mammalian cells, LA is reduced to dihydrolipoic acid (DHLA), a vicinal dithiol, and rapidly effluxed into the extracellular milieu. We hypothesized that DHLA may be an effective inhibitor of human gelatinase B (GelB). Purified human GelB was incubated with 0 to 200 micromol/L DHLA, and residual enzyme activity was measured by HPLC using a fluorogenic substrate (matrix metalloproteinase substrate III). DHLA inhibited GelB in a dose-dependent fashion with an IC50 of 20 micromol/L. Oxidation of DHLA resulted in a loss of DHLA's capacity to inhibit GelB. The DHLA-mediated inhibition of GelB was independent of the zinc concentration in the reaction buffer. DHLA had no inhibitory effect on gelatinase A. Zymographs of activated neutrophil lysates demonstrated that higher concentrations of DHLA also prevent the activation of GelB proenzyme. Bronchoalveolar lavage fluid from mice fed a diet enriched with LA showed significantly increased GelB inhibitory capacity (p = 0.0002 vs. regular diet). We conclude that DHLA can modulate neutrophil-derived GelB activity through direct inhibition of enzyme activity and by preventing the activation of GelB proenzyme.

Knowledge on molecular systems involved in myogenic precursor cell (mpc) fusion into myotubes is fragmentary. Previous studies have implicated the a disintegrin and metalloproteinase (ADAM) family in most mammalian cell fusion processes. ADAM12 is likely involved in fusion of murine mpc and human rhabdomyosarcoma cells, but it requires yet unknown molecular partners to launch myogenic cell fusion. ADAM12 was shown able to mediate cell-to-cell attachment through binding alpha9beta1 integrin. We report that normal human mpc express both ADAM12 and alpha9beta1 integrin during their differentiation. Expression of alpha9 parallels that of ADAM12 and culminates at time of fusion. alpha9 and ADAM12 coimmunoprecipitate and participate to mpc adhesion. Inhibition of ADAM12/alpha9beta1 integrin interplay, by either ADAM12 antisense oligonucleotides or blocking antibody to alpha9beta1, inhibited overall mpc fusion by 47-48%, with combination of both strategies increasing inhibition up to 62%. By contrast with blockade of vascular cell adhesion molecule-1/alpha4beta1, which also reduced fusion, exposure to ADAM12 antisense oligonucleotides or anti-alpha9beta1 antibody did not induce detachment of mpc from extracellular matrix, suggesting specific involvement of ADAM12-alpha9beta1 interaction in the fusion process. Evaluation of the fusion rate with regard to the size of myotubes showed that both ADAM12 antisense oligonucleotides and alpha9beta1 blockade inhibited more importantly formation of large (> or =5 nuclei) myotubes than that of small (2-4 nuclei) myotubes. We conclude that both ADAM12 and alpha9beta1 integrin are expressed during postnatal human myogenic differentiation and that their interaction is mainly operative in nascent myotube growth.

Elastin-derived peptides display a wide range of biological activities in a number of normal and transformed cells but their involvement in angiogenesis has not been reported. In the present study, we show that kappa-elastin and VGVAPG hexapeptide elastin motif accelerated angiogenesis in the chick chorio-allantoic membrane in an in vivo model. They also stimulated pseudotube formation from human vascular and microvascular endothelial cells in the matrigel and collagen models as well as cell migration in an in vitro wound healing assay. Confocal and scanning electron microscopy analyses revealed the main reorganization of actin filaments mediated by elastin-derived peptides and changes in cell shape that correlated with a decrease of the cell form factor determined by computerized image analysis. Such elastin-derived peptide effects were attributed to upregulation of proMT1-MMP and proMMP-2 expression and activation at both the mRNA and protein levels. Batimastat, an inhibitor of furin convertase and TIMP-2, but not TIMP-1, totally abolished the influence of elastin-derived peptides (EDPs) on cell migration and tubulogenesis, thus favoring the involvement of MT1-MMP in such processes. To assess its contribution to EDP-mediated angiogenesis further, we used a small interfering RNA (siRNA) approach for specifically silencing MT1-MMP in human microvascular endothelial cells. Four sets of 21 bp siRNA duplexes targeting MT1-MMP mRNA were synthesized by in vitro transcription. Two of them proved to inhibit MT1-MMP expression efficiently but did not affect MT2-, MT3- and MT5-MMP expression. Seventy-two hours after transfection with 25 nM siRNAs EDP-induced MT1-MMP expression at the mRNA and protein levels was decreased fourfold. In parallel, proMMP-2 activation was inhibited. A scrambled siRNA, used as a negative control, had no effect. Finally, the effect of elastin peptides on pseudotube formation in MT1-MMP-siRNA transfected cells was totally abolished. These data emphasise the crucial role of MT1-MMP in the elastin-induced angiogenic phenotype of endothelial cells.

We have investigated the physiological function of type 2 methionine aminopeptidases (MetAP2) using Caenorhabditis elegans as a model system. A homolog of human MetAP2 was found in the C. elegans genome, which we termed MAP-2. MAP-2 protein displayed methionine aminopeptidase activity and was sensitive to inhibition by fumagillin. Downregulation of map-2 expression by RNAi led to sterility, resulting from a defect in germ cell proliferation. These observations suggest that MAP-2 is essential for germ cell development in C. elegans and that this ubiquitous enzyme may play important roles in a tissue specific manner., (Copyright 2004 Federation of European Biochemical Societies)

UVA exposure causes skin photoaging by singlet oxygen (1)O(2)-mediated induction of, e.g., matrix metalloproteases (MMPs). We assessed whether pretreatment with beta-carotene, a (1)O(2) quencher and retinoic acid (RA) precursor, interferes with UVA-induced gene regulation. HaCaT keratinocytes were precultured with beta-carotene at physiological concentrations (0.5, 1.5, and 3.0 microM) prior to exposure to UVA from a Hönle solar simulator (270 kJ/m(2)). HaCaT cells accumulated beta-carotene in a time- and dose-dependent manner. UVA irradiation massively reduced the cellular beta-carotene content. Beta-carotene suppressed UVA-induction of MMP-1, MMP-3, and MMP-10, three major matrix metalloproteases involved in photoaging. We show that regulation by not only MMP-1, but also MMP-10, involves (1)O(2)-dependent mechanisms. Beta-carotene dose-dependently quenched (1)O(2)-mediated induction of MMP-1 and MMP-10. Thus, as in chemical solvent systems, beta-carotene quenches (1)O(2) also in living cells. Vitamin E did not cooperate with beta-carotene to further inhibit MMP induction. HaCaT cells produced weak retinoid activity from beta-carotene, as demonstrated by mild upregulation of RAR beta and activation of an RARE-dependent reporter gene. Beta-carotene did not regulate the genes encoding other RARs, RXRs, or the two beta-carotene cleavage enzymes. These results demonstrate that beta-carotene acts photoprotectively, and that this effect is mediated by (1)O(2) quenching.

Human adamalysin 19 (a disintegrin and metalloproteinase 19, hADAM19) is activated by furin-mediated cleavage of the prodomain followed by an autolytic processing within the cysteine-rich domain at Glu586-Ser587, which occurs intramolecularly, producing an NH2 terminal fragment (N-fragment) associated with its COOH-terminal fragment (C-fragment), most likely through disulfide bonds. When stable Madin-Darby canine kidney (MDCK) transfectants overexpressing soluble hADAM19 were treated with dithiothreitol (DTT) or with media at pH 6.5, 7.5, or 8.5, the secretion and folding of the enzyme were not affected. Autolytic processing was blocked by DTT and pH 6.5 media, which favor disulfide reduction, but was increased by pH 8.5 media, which promotes disulfide formation. Cys605, Cys633, Cys639, and Cys643 of the C-fragment appear to be partially responsible for the covalent association between the C-fragment and the N-fragment. A new autolytic processing site at Lys543-Val544 was identified in soluble mutants when these cysteine residues were individually mutated to serine residues. Shed fragments were also detectable in the media from MDCK cells stably expressing the full-length Cys633Ser mutant. Ilomastat/GM6001 inhibited hADAM19 with an IC50 of 447 nM, but scarcely affected the shedding process. The cysteine-rich domain likely forms disulfide bonds to regulate the autolytic processing and shedding of hADAM19.

Matrix metalloproteinases (MMPs) and TNF-alpha converting enzyme (TACE) contribute to the pathophysiology of bacterial meningitis. To date, MMP-inhibitors studied in models of meningitis were compromised by their hydrophobic nature. We investigated the pharmacokinetics and the effect of TNF484, a water-soluble hydroxamate-based inhibitor of MMP and TACE, on disease parameters and brain damage in a neonatal rat model of pneumococcal meningitis. At 1 mg/kg q6h TNF484 reduced soluble TNF-alpha and the collagen degradation product hydroxyproline in the cerebrospinal fluid. Clinically, TNF484 attenuated the incidence of seizures and was neuroprotective in the cortex. Water-soluble MMP-inhibitors may hold promise in the therapy of bacterial meningitis.

Cigarette smoke (CS) causes pulmonary emphysema in humans and elastin degradation plays a key role in its pathogenesis. Previous studies on CS-exposed animals have been equivocal and have not clearly demonstrated the progression of the disease. In this study, morphometry was used to assess lung modifications to alveolar septa, airspaces, elastic and collagen fibers, and alveolar macrophages. Male (n = 40) C57/BL6 mice were exposed 3 times/day, whole body, to CS from three cigarettes for 10, 20, 30, or 60 days. Control groups (n = 10) were sham-smoked or received no exposure (day 0, n = 10). Morphometry included measurements of volume fraction of alveolar septa and airspaces, elastic and collagen fibers, and surface fraction of elastic fibers and alveolar septa. Morphometrical differences in mice after 60 days of exposure were greater than those after 10, 20, or 30 days, suggesting a progression of the disease. Inflammatory lesions in the lungs of mice contained significantly more metalloelastase (MMP-12) in macrophages at 10, 20, and 30 days than in controls of mice exposed for 60 days. These results suggest that elastin degradation took place during development of pulmonary changes in mice exposed to CS, and activation of MMPs specific for elastin may be a determining factor for susceptibility to emphysema.

TNP-470, the first anti-angiogenic small molecule to enter clinical trials, targets methionine aminopeptidase-2 (MetAP-2), a metalloprotease that cleaves the N-terminal methionine of proteins. Previously, biochemical binding, in vivo yeast studies, and structural studies of human methionine aminopeptidase-2 bound to TNP-470 and its analogs fumagillin and ovalicin revealed that these compounds exhibit specificity for MetAP-2 over its family member MetAP-1. To further elucidate the nature of this specificity, we developed a yeast-based screen for human MetAP-2 mutations that confer ovalicin resistance. Of the three resistant alleles, A362T appeared in the majority of clones and was found to be the most resistant to the ovalicin class of inhibitors. Alignment of human MetAP-2 with human MetAP-1, which is naturally ovalicin-resistant, revealed that the analogous residue in MetAP-1 is also a threonine. Mutation of this residue to alanine resulted in an ovalicin-sensitive MetAP-1 allele, demonstrating that an alanine at this position is critical for inhibition by ovalicin. These results provide a molecular explanation for the specificity exhibited by this class of anti-angiogenic agents for MetAP-2 over MetAP-1 and may prove useful in the development of additional MetAP-2-specific therapeutic agents.

The PPAR gamma agonists, thiazolidinediones (TZDs), have anti-inflammatory properties as well as increasing insulin sensitivity. This has widened their therapeutic scope to treat inflammatory diseases such as atherosclerosis in addition to Type 2 Diabetes. TZDs are known to reduce monocyte/macrophage expression of Matrix metalloproteinase (MMP)-9, which is implicated in atherosclerotic plaque destabilization. This study aims to identify other metalloproteinase genes of the ADAM (A Disintegin And Metalloproteinase) and ADAMTS families that are regulated by PPAR gamma or RXR agonists, which are potentially important in type 2 diabetes and/or related atherosclerosis. The synthetic PPAR gamma agonist, GW7845, and the natural agonist 15d-PGJ2, suppressed PMA stimulated MMP-9 in human monocyte-like cells (THP-1) only in the presence of 9-cis-retinoic acid. Quantitative Real-Time PCR showed that this reduction was regulated at the mRNA level. Expression of ADAMs 8, 9, and 17 were increased, and ADAM15 was decreased by stimulation of THP-1 with PMA, although these ADAMs were not regulated by PPAR gamma or RXR agonists. PMA-induced ADAM28 expression was further enhanced by the addition of 9-cis-retinoic acid. ADAMTS4, implicated in rheumatoid arthritis, was expressed in THP-1 cells, and significantly increased after 24 h of PMA stimulation. ADAMTS4 expression was suppressed by both PPAR gamma and RXR agonists and was undetectable when the agonists were combined. Pretreatment of THP-1 cells with the PPAR gamma antagonist, GW9662, suggests that PPAR gamma plays subtly different roles in the regulation of MMP-9, ADAMTS4 and ADAM28 gene expression. These results indicate that PPAR gamma and RXR agonists have complex effects on monocyte metalloproteinase expression, which may have implications for therapeutic strategies.

Radiation therapy is a widely-used option for the treatment of a variety of solid tumors. Although effective, ionizing radiation (IR) may give rise to various side effects, including secondary tumors. In agreement with this, recent reports have demonstrated increased invasive potential in different tumor-derived cell lines following radiation treatment. Many of the molecular effects of IR specifically on the endothelial cells involved in tumor neo-vascularization remain unknown. In this study, we found that low sublethal single doses of IR applied to human umbilical vein endothelial cells stimulated cell migration and in vitro tubulogenesis. This correlated with an increase in membrane type-1 matrix metalloproteinase (MT1-MMP) protein expression, a crucial enzyme that promotes endothelial cell migration and tube formation, and of caveolin-1, a protein that regulates tube formation. Cell adhesion was also promoted by IR, reflected in increased gene expression levels of cell surface beta(3) integrin. Pretreatment of the cells with epigallocatechin-3-gallate (EGCg), a green tea catechin that possesses anti-angiogenic properties, prevented most of the IR-induced cellular and molecular events. These observations suggest that current protocols involving radiation therapy for the treatment of cancer can paradoxically promote angiogenesis, but can be improved by combination with anti-angiogenic molecules such as EGCg to target those tumor-derived endothelial cells that escaped IR-induced apoptosis.

Understanding the reaction mechanism of co-catalytic metallopeptidases provides a starting point for the design and synthesis of new molecules that can be screened as potential pharmaceuticals. Many of the enzymes that contain co-catalytic metallo-active sites play important roles in cellular processes such as tissue repair, protein maturation, hormone level regulation, cell-cycle control and protein degradation. Therefore, these enzymes play central roles in several disease states including cancer, HIV, stroke, diabetes, bacterial infections, neurological processes, schizophrenia, seizure disorders, and amyotrophic lateral sclerosis. The mechanism of AAP, an aminopeptidase from Aeromonas proteolytica, is one of the best-characterized examples of a metallopeptidase containing a co-catalytic metallo-active site, although this enzyme is not a specific pharmaceutical target at this time. As a large majority of co-catalytic metallopeptidases contain active sites that are nearly identical to the one observed in AAP, the major steps of their catalytic mechanisms are likely to be very similar. With this in mind, it is possible to propose a general catalytic mechanism for the hydrolysis of amino acid substrates.

Objective: This report describes the experience of a case of atypical thrombotic thrombocytopenic purpura (TTP) whose diagnosis was based on severe deficiency of the von Willebrand factor (vWF) cleaving metalloprotease ADAMTS13., Methods: The level of ADAMTS13 activity, the titer of the inhibitors of this protease and the size distribution of vWF multimers in plasma samples were analysed in a patient with recurrent episodes of dizziness and blurred vision., Results: In the absence of thrombocytopenia or microangiopathic hemolysis, diagnosis of TTP was established by demonstration of very low ADAMTS13 activity levels and the presence of inhibitors of this protease. After rituximab therapy decreased the inhibitor titer and increased the ADAMTS13 level, the patient has had no relapse of ischemic symptoms in the following 16 months., Conclusions: Acute neurological deficits may occur in TTP without concurrent thrombocytopenia or microangiopathic hemolysis. The role of rituximab for patients with TTP deserves further exploration.

von Willebrand factor-cleaving protease (ADAMTS13) cleaves von Willebrand factor (VWF) and regulates its physiologic function. To investigate the relation between ADAMTS13 activity and VWF, we compared ADAMTS13 activity with the VWF-related parameters VWF antigen (VWF:Ag), VWF collagen-binding activity (VWF:CBA), VWF-propeptide, proVWF, and VWF multimeric composition in 10 healthy volunteers and 3 patients with type 1 von Willebrand disease before and after infusing 0.3 microg/kg desmopressin. The VWF-related parameters in the volunteers increased 60 minutes after start of infusion by 3.7-fold for VWF:Ag, 7.2-fold for propeptide, and 2.2-fold for VWF:CBA. Unusually large VWF multimers and traces of proVWF appeared. The ADAMTS13 activity decreased to about half the initial value. After 24 hours values returned to baseline. Patients with type 1 von Willebrand disease showed similar results. We conclude that the inverse correlation between ADAMTS13 and VWF-related parameters suggests a consumption of ADAMTS13 after the desmopressin-induced release of higher multimers of VWF.

The reprolysin subfamily of metalloproteinases includes snake venom metalloproteinases (SVMP) and mammalian disintegrin/metalloproteinase. These proteins are synthesized as zymogens and undergo proteolytic processing resulting in a variety of multifunctional proteins. Jararhagin is a P-III SVMP isolated from the venom of Bothrops jararaca. In crude venom, two forms of jararhagin are typically found, full-length jararhagin and jararhagin-C, a proteolytically processed form of jararhagin that is composed of the disintegrin-like and cysteine-rich domains of jararhagin. To better understand the structural and mechanistic bases for these forms of jararhagin in the venom of B. jararaca and the source of venom complexity in general, we have examined the jararhagin forms isolated from venom and the autolysis of isolated jararhagin under the conditions of varying pH, calcium ion concentration, and reducing agents. From our results, jararhagin isolated from venom appears as two forms: a predominant form that is stable to in vitro autolysis and a minor form that is susceptible to autolysis under a variety of conditions including alkaline pH, low calcium ion concentrations, or reducing agent. The autolysis site for production of jararhagin-C from isolated jararhagin was different from that observed for jararhagin-C as isolated from crude venom. Taken together, these data lead us to the conclusion that during the biosynthesis of jararhagin in the venom gland at least three forms are present: one form which is rapidly processed to give rise to jararhagin-C, one form which is resistant to processing in the venom and autolysis in vitro, and one minor form which is susceptible to autolysis under conditions that promote destabilization of its structure. The presence of these different forms of jararhagin contributes to greater structural and functional complexity of the venom and may be a common feature among all snake venoms. The biological and biochemical features in the venom gland responsible for these jararhagin isoforms are currently under investigation.

Proteomic analysis is an important approach to characterize the proteome and study protein functions. It is also a powerful screening method for detecting unexpected alterations in protein expression that may be overlooked by conventional biochemical techniques. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) is an alkylating agent that can induce nontargeted mutagenesis in treated cells, although the mechanism remains unclear. Using an efficient proteomic method, we identified several cellular proteins that are responsive to low-concentration MNNG treatment in human FL cells. After MNNG treatment, whole cellular proteins were separated using two-dimensional gel electrophoresis and visualized by silver staining; the digitized images then were analyzed with 2D analysis software. More than 60 proteins showed significant changes in MNNG-treated cells compared to control cells (DMSO treatment). Thirty-one proteins only detected in MNNG-treated or control cells were subjected to in-gel digestion with trypsin and identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry using peptide mass fingerprinting. Eighteen of theses proteins have been identified, including several zinc finger proteins, two members of the ADAMs (a disintegrin and metalloprotease domain) family, and two integrins. Most of these proteins have unknown functions and their involvement in the cellular responses to alkylating agents have not been reported. Therefore, our findings may offer new insights into the mechanisms of low-concentration MNNG-induced nontargeted mutagenesis and these proteins may serve as new biomarkers for detecting exposure of human populations to environmental carcinogens., (Copyright 2003 Wiley-Liss, Inc.)

The aim of this study is to explain the selectivity of the antiangiogenic drug fumagillin for the eukaryotic enzyme methionine aminopeptidase type II (MetAP-II, EC 3.4.11.18) over the structurally very similar MetAP-I. A homology model for the human MetAP-I is constructed and molecular dynamics simulations are performed on this model with and without a docked fumagillin molecule. These simulations are compared with analogous simulations that were performed on the experimentally determined structure of the human MetAP-II enzyme. We observe an increased flexibility of the active site histidine that is covalently modified by fumagillin in the MetAP-I enzyme. The MetAP-I active site residues, particularly the fumagillin-binding histidine, have a lower probability to be in a conformation that is prone to react with the drug than their MetAP-II counterparts. This result offers an explanation for the selectivity of fumagillin for the eukaryotic MetAP-II enzyme.

Enamel fluorosis is characterised by increased porosity and a delay in the removal of enamel matrix proteins as the enamel matures. Amelogenin is the primary matrix protein in secretory-stage dental enamel. As enamel matures, amelogenins are hydrolysed by a number of enamel proteinases, including matrix metalloproteinase-20 (MMP-20 or enamelysin) and serine proteinase. Here, the effect of ingested fluoride on the relative activity of proteinases in the enamel matrix and the specific effect of fluoride on MMP-20 activity were examined. Proteinase activity relative to total enamel matrix protein was measured by fluorescence assay of enamel matrix dissected from rats given 0, 50, or 100 parts per 10(6) fluoride in their drinking water. To determine the specific effect of fluoride on the activity of MMP-20, the hydrolysis of a full-length recombinant human amelogenin by recombinant MMP-20 (rMMP-20) in the presence of 0, 2, 5, 10 or 100 microM fluoride was compared by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE). In addition, a fluorescent peptide assay was developed to quantify enzyme activity against the tyrosine-rich amelogenin peptide cleavage site. In the late maturation stage, total proteinase activity per unit protein was lower in the fluoride-exposed rats than in the control rats. This in vivo finding indicates that fluoride ingestion can alter the relative amount of active proteinase in mature enamel. Hydrolysis of amelogenin at neutral pH by rMMP-20 was reduced in the presence of 100 microM F. In the peptide assay, rMMP-20 activity was significantly reduced by concentrations of fluoride as low as 2 microM at pH 6, with no significant effect at pH 7.2. These in vitro assays show that micromolar concentrations of fluoride can alter metalloproteinase activity, particularly when the pH is reduced to 6.0. These studies suggest that the effects of fluoride on enamel matrix proteinase secretion or activity could be involved in the aetiology of fluorosis in enamel and other mineralising tissues.

While most patients of thrombotic thrombocytopenic purpura (TTP) respond to plasma exchange and achieve remission, a subset of the patients experience multiple relapses or develop a persistent disease that may be debilitating and life threatening. We report the experience of rituximab treatment in three consecutive patients who had required periodic plasma exchange for greater than 50-180 days after failing other modalities of treatments. Two patients each received eight doses of rituximab infusion and had clinical remissions that have lasted for 23 months and ongoing in one patient and for 17 months before relapse in the other. The third patient received four doses of rituximab infusion and had improvement of disease for 60 days until her death from unrelated causes. Analysis of the von Willebrand factor-cleaving metalloprotease activity (ADAMTS13) and its inhibitor in two patients showed that rituximab treatment was associated with a rise of the protease level and a decrease of the inhibitor titers in one patient who achieved clinical remission and a decrease of the inhibitor titer but no increase in protease level in the other patient who had a partial response. Based on the responses to rituximab and its mild toxicity in these three patients, and the lack of effective alternative treatments, additional exploration of the role of rituximab in the treatment of refractory TTP is warranted.

Oxidized low-density lipoprotein (oxLDL), which accumulates in vascular lesions, alters vascular cell function in ways that can be construed as atherogenic. Among these is the observation that oxLDL and its lipids promote smooth muscle cell (SMC) proliferation. A number of schemes have been proposed to explain this phenomenon. Our published data support the concept that part of the proliferation is mediated by lysophosphatidylcholine (lysoPC) and structurally related phospholipids borne by oxLDL, which cause FGF-2 release via an oxidant-dependent mechanism. Since FGF-2 can bind extracellular matrices, we wanted to determine whether the FGF-2 released came from an intracellular or an extracellular matrix-bound pool. We tested whether lysoPC was capable of releasing FGF-2 from SMC matrices, whether agents that release FGF-2 from matrices could cause proliferation, and whether lysoPC-mediated proliferation could occur by stimulating metalloproteinase (MMP)-induced matrix degradation, which released matrix-bound FGF-2. Our results indicate that the source of FGF-2 released by lysoPC and related lipids is a preexisting cellular pool and not from matrix, and that the mechanism likely involves transient, sublethal cell permeabilization. These results enhance understanding of a mechanism by which oxLDL could contribute to SMC proliferation in arterial lesions.

Aims: intimal hyperplasia is mediated by smooth muscle cell proliferation, migration and deposition of extracellular matrix. The anti-fibrotic agent pirfenidone has been shown to inhibit pro-fibrotic growth factors in non-vascular inflammatory models. This study investigated the effect of the novel anti-fibrotic agent pirfenidone on the development of neointima., Methods: male Sprague-Dawley rats received either standard diet or diet supplemented with pirfenidone (250, 500, 1000 mg/kg/day). Animals underwent left common carotid balloon angioplasty and were explanted at 4, 8, 14 and 28 days and analysed for intimal thickening, pro-fibrotic gene expression, extracellular matrix deposition and metalloproteinase activity., Results: neointimal thickness was significantly reduced in a dose-dependent manner at 14 days; pirfenidone 250 mg/kg (p<0.005), pirfenidone 500 mg/kg (p<0.001), pirfenidone 1 g/kg ( p<0.001). There were no significant differences in intimal thickening at 28 days. Expression of MMP-2, MMP-9, TIMP-1, collagen III and TGF-beta were all significantly inhibited at 14 days. Both collagen III expression and ECM deposition were reduced at 28 days ( p<0.05 and <0.002 respectively)., Conclusion: pirfenidone reduces expression of MMPs governing smooth muscle cell proliferation and migration (MMP-2 and 9), and genes favouring ECM accumulation (TIMP-1 and collagen III). This study shows that inhibition of MMP activity is not sufficient to inhibit late lesion size., (Copyright 2002 Harcourt Publishers Limited.)

We report herein the case of a 69-year-old Japanese man with acquired von Willebrand syndrome associated with monoclonal gammopathy of undetermined significance who developed IgG1-kappa antibodies against von Willebrand factor (VWF). The patient was urged to undergo tooth extractions because of alveolar pyorrhea, and a low-dosage intravenous immunoglobulin (IV-Ig) therapy (0.3 g of IgG/kg/day for 3 days) was chosen for him. On the 4th day after the infusion, VWF antigen and VWF ristocetin cofactor increased to 40 and 78% of the control, respectively, and dental extractions were performed successfully. On the 7th day, these values reached a maximum, i.e. 95 and 160% of the control, respectively. Then, they quickly decreased to 35 and 75% on the 10th day, and 6 months later, they became 16 and <3% of the control, respectively. Upon analysis of plasma VWF multimers (VWFMs) in this patient, those with large to medium molecular masses more selectively disappeared before the IV-Ig infusion than did those with small molecular masses. On the 4th day, the pattern of VWFMs was completely normalized and appeared to persist until the 10th day. Six months later, a small amount of large to medium-sized VWFMs was still present, but at 7-8 months, the pattern of VWFMs became almost the same as that before infusion. Throughout the patient's clinical course, the activity of plasma VWF-cleaving protease, which specifically cleaves the Tyr842-Met843 bond of the subunit and reduces its multimeric sizes, was quite normal (95-119%). These results provided consistent evidence that the selective absence of VWFMs with large to medium molecular masses in this patient is caused by the heightened clearance of a complex of IgG inhibitor and VWFMs from the circulation, presumably through IgG binding to the Fc receptor of macrophages. Furthermore, these results also indicated that a low-dosage IV-Ig therapy is effective enough for hemostatic management for programmed surgery., (Copyright 2002 S. Karger AG, Basel)

During testicular development and maturation, extracellular matrix (ECM) remodelling is a fundamental process which requires the presence of several proteases and protease inhibitors. Among the proteases, a pivotal role has been proposed for matrix metalloproteases (MMPs) and their tissue inhibitors (TIMPs). Here we report an analysis of MMP-2, MT1-MMP and TIMP-2 expression by rat Sertoli cells in culture using RT-PCR and zymographic techniques. Stimulating Sertoli cells with follicle-stimulating hormone (FSH) in vitro induced evident changes in the level of their mRNA in a time-dependent manner. In the case of TIMP-2 and MT1-MMP, the respective transcripts were augmented up to three-fold after 24 h of treatment, and MMP-2 transcripts increased by four times in the same period. MMP-2 activity determined by gelatin zymography showed an increase in enzyme secretion after FSH stimulation. The results of this study suggest that: (i) at the surface of Sertoli cells pro-MMP-2 activation mediated by MT1-MMP may occur, involving TIMP-2 as a receptor; and (ii) the expression of these molecules is not constitutive in this cell type, but may be modulated by FSH and is therefore implicated in spermatogenesis.

Platelet Activating Factor (PAF), an inflammatory bioactive lipid, has been shown to be involved in the regulation of the activity of matrix metalloproteinases (MMPs). In view of the role played by MMPs in tumor cell invasiveness, we investigated whether PAF influences MMP activity in a system of neuroblastoma clones, the AA5 and AE12 cells, isolated from the human LaN1 neuroblastoma cell line. These clones were characterized by an inverse relationship between invasiveness and differentiative capacity and by the expression of specific cell surface PAF receptors. We found that the levels of mRNAs specific for MMP-2 and for MT1-MMP, the MMP-2 activator, were reduced in both clones treated with 300 nM PAF. These changes are consistent with the reduced secretion and activation of MMP-2 found in the neuroblastoma clones exposed to PAF. These effects were accompanied by an inhibition of invasiveness through Matrigel and by a promotion of differentiation, as revealed by an increased percentage of cells with neurites. The finding that both neuroblastoma clones exposed to the metalloproteinase inhibitors, BB3103 and 1,10-phenanthroline, increased their differentiative capacity and reduced their invasiveness through Matrigel, represents a further indication that PAF modulates differentiation and invasiveness by affecting the activity of MMPs.

Background: Hepatic fibrosis occurs because of injury to the liver parenchyma and biliary system. We have investigated the effect of an organic selenium anti-oxidant, ebselen, in the resolution of experimentally induced hepatic fibrosis, and evaluated its effect on various paradigms involved in hepatic fibrosis., Methods: Following pretreatment with phenobarbitone, liver fibrosis was induced in male Fischer 344 rats by using carbon tetrachloride treatment for 10 weeks. Carbon tetrachloride-treated rats were randomly assigned into two groups: (i) no ebselen; and (ii) ebselen administered for 3 weeks following a 10-week carbon tetrachloride treatment period. Normal controls were: (i) neither carbon tetrachloride nor ebselen treated; or (ii) ebselen treated for 13 weeks. Liver sections were stained with hematoxylin and eosin, Masson trichrome and stained for reticulin by using silver impregnation. Reverse transcription-polymerase chain reaction was used to analyze the steady-state levels of gene(s) involved in: (i) hepatic fibrosis, namely, transforming growth factor-beta1, procollagen I and III, tissue inhibitor of metalloproteinase-1 and matrix metalloproteinase-13; (ii) oxidative stress, namely, cytochrome P4502E1; and (iii) preneoplastic liver foci, namely, the placental form of glutathione-S-transferase., Results: Histological staining showed that ebselen resolves carbon tetrachloride-induced hepatic fibrosis. Treatment with ebselen reduced steady-state levels of transforming growth factor-beta1, procollagen I and III, tissue inhibitor of metalloproteinase-1, cytochrome P4502E1 and placental form glutathione-S-transferase transcripts, and increased transcripts of matrix metalloproteinase-13., Conclusion: These findings provide evidence that ebselen significantly causes the resolution of carbon tetrachloride-induced hepatic fibrosis in rats.

It has been previously demonstrated that aspartic, serine, metallo and cysteine proteases bind to their inhibitors and substrate analogues in a single conformation, the saw-tooth or extended beta-strand. Consequently a generic approach to the development of protease inhibitors is the use of constraints that conformationally restrict putative inhibitor molecules to an extended form. In this way the inhibitor is pre-organized for binding to a protease and does not need to rearrange its structure. One constraining device that has proven to be effective for such pre-organization is macrocyclization. This article illustrates the general principle that macrocycles, especially those composed of 3-4 amino acids and usually 13-17 ring atoms, can effectively mimic the extended conformation of short peptide sequences. Such structure-stabilising macrocycles are stable to degradation by proteases, valuable components of potent protease inhibitors, and in many cases they are also bioavailable.

Tumour necrosis factor-alpha (TNF-alpha) is a major immunomodulatory and proinflammatory cytokine which is shed in its soluble form by a membrane-anchored zinc protease, identified as a disintegrin and metalloproteinase (ADAM) called TNF-alpha convertase (TACE; ADAM17). The role of this protease in the adult nervous system remains poorly understood. During cerebral ischemia and subsequent reperfusion, expression and release of TNF-alpha have been shown. We have investigated the expression and activity of TACE in an in vitro model of brain ischemia consisting of rat forebrain slices exposed to oxygen-glucose deprivation (OGD). OGD caused the release of TNF-alpha, an effect which was inhibited by a hydroxamate-based metalloprotease inhibitor, BB-3103, with an IC(50) of 0.1 microM, suggesting that TNF-alpha release results selectively from TACE activity. Assay of TACE enzymatic activity on a fluorescein-labelled peptide spanning the cleavage site in pro-TNF-alpha, as well as Western blot and RT-PCR analyses showed that TACE is present in control forebrain and, more interestingly, that TACE expression is increased in OGD-exposed tissue. TACE enzymatic activity from OGD-exposed slices was significantly inhibited by cycloheximide, suggesting that de novo synthesis of TACE contributes to TNF-alpha release after ischaemia. Moreover, it was also inhibited by bisindolylmaleimide I, indicating that TACE activity is regulated by PKC. These findings posed the question of what was its function therein. Among other actions, TNF-alpha has been described to be involved in the expression of inducible nitric oxide synthase (iNOS), a high-output NOS isoform associated to cellular damage, but the link between TNF-alpha release after brain ischaemia and iNOS expression in this condition has not been shown. We have now found that iNOS expression in OGD-subjected brain slices is inhibited by BB-3103 at concentrations below 1 microM, indicating that shedding of TNF-alpha by TACE plays a necessary part in the induction of this NOS isoenzyme after OGD. Taken together, these data demonstrate that (1) TACE/ADAM17 activity accounts for the majority of TNF-alpha shedding after OGD in rat forebrain slices, (2) an increase in TACE expression contributes, at least in part, to the rise in TNF-alpha after OGD and (3) iNOS expression in OGD-subjected brain slices results from TACE activity and subsequent increase in TNF-alpha levels.

Flavobacterium psychrophilum is a fish pathogen that commonly affects salmonids. This bacterium produced an extracellular protease with an estimated molecular mass of 55 kDa. This enzyme, designated Fpp1 (F. psychrophilum protease 1), was purified to electrophoretic homogeneity from the culture supernatant by using ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography, and size exclusion chromatography. On the basis of its biochemical characteristics, Fpp1 can be included in the group of metalloproteases that have an optimum pH for activity of 6.5 and are inhibited by 1,10-phenanthroline, EDTA, or EGTA but not by phenylmethylsulfonyl fluoride. Fpp1 activity was dependent on calcium ions not only for its activity but also for its thermal stability. In addition to calcium, strontium and barium can activate the protein. The enzyme showed typical psychrophilic behavior; it had an activation energy of 5.58 kcal/mol and was more active at temperatures between 25 and 40 degrees C, and its activity decreased rapidly at 45 degrees C. Fpp1 cleaved gelatin, laminin, fibronectin, fibrinogen, collagen type IV, and, to a lesser extent, collagen types I and II. Fpp1 also degraded actin and myosin, basic elements of the fish muscular system. The presence of this enzyme in culture media was specifically dependent on the calcium concentration. Fpp1 production started early in the exponential growth phase and reached a maximum during this period. Addition of calcium during the stationary phase did not induce Fpp1 production at all. Besides calcium and the growth phase, temperature also seems to play a role in production of Fpp1. In this study we found that production of Fpp1 depends on factors such as calcium concentration, growth phase of the culture, and temperature. The combination of these parameters corresponds to the combination in the natural host during outbreaks of disease caused by F. psychrophilum. Consequently, we suggest that environmental host factors govern Fpp1 production.

The activation of progelatinase A to gelatinase A requires cleavage of an asparaginyl bond to form the N-terminus of the mature enzyme. We have asked whether the activation can be mediated by legumain, the recently discovered lysosomal cysteine proteinase that is specific for hydrolysis of asparaginyl bonds. Addition of purified legumain to the concentrated conditioned medium from HT1080 cell culture that contained both progelatinases A and B caused the conversion of the 72 kDa progelatinase A to the 62 kDa form. The progelatinase B in the medium was unaffected. Incubation of recombinant progelatinase A with legumain resulted in an almost instantaneous activation as judged by the fluorometric assay with a specific gelatinase A substrate, Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2. Legumain also activated progelatinase A when it was in complex with TIMP-2. Zymographic analysis and N-terminal sequencing revealed that legumain cleaved the 72 kDa progelatinase A at the bonds between Asn109-Tyr110 or Asn111-Phe112 to produce the 62 kDa mature enzyme, and that further cleavage at Asn430 also occurred to generate a 36 kDa active form. More 62 kDa gelatinase A was detected in cultures of C13 cells that over-expressed legumain than in those of the control HEK293 cells. We conclude that legumain is clearly capable of processing progelatinase A to the active enzyme in vitro and in cultured cells.

The PST-01 protease is secreted by the organic solvent-tolerant microorganism Pseudomonas aeruginosa PST-01 and is stable in the presence of various organic solvents. Therefore, the PST-01 strain and the PST-01 protease are very useful for fermentation and reactions in the presence of organic solvents, respectively. The organic solvent-stable PST-01 protease has two disulfide bonds (between Cys-30 and Cys-58 and between Cys-270 and Cys-297) in its molecule. Mutant PST-01 proteases in which one or both of the disulfide bonds were deleted were constructed by site-directed mutagenesis, and the effect of the disulfide bonds on the activity and the various stabilities was investigated. The disulfide bond between Cys-270 and Cys-297 in the PST-01 protease was found to be essential for its activity. The disulfide bond between Cys-30 and Cys-58 played an important role in the organic solvent stability of the PST-01 protease.

The angiogenic action of thrombin has been shown to be mediated by activation of the thrombin receptor. In this report we studied the effects of SFLLR, an agonist of the activated thrombin receptor and thrombin receptor peptide and non peptide antagonists on angiogenesis in the chick chorioallantoic membrane (CAM) system. As antagonists were used the tripeptide FPR and non-peptide 1,4-disubstituted piperazine derivatives. The pentapeptide SFLLR, like thrombin, caused a marked stimulation of angiogenesis in the CAM. FPR and the piperazine derivatives caused suppression of angiogenesis and in combination with thrombin antagonized its angiogenic effect. Thrombin and SFLLR activated progelatinase A (MMP-2) in the culture medium of human umbilical cord endothelial cells (HUVECs). MMP-2 is involved in the early steps of angiogenesis leading to local dissolution of basement membrane collagen and migration of the activated endothelial cells. FPR and the piperazine derivatives inhibited the activation of this enzyme. They also antagonised the effects of both thrombin and SFLLR on MMP-2 activation. These results suggest that non-thrombogenic agonists or antagonists of the activated thrombin receptor can be used as modulators of angiogenesis.

The desmoplastic response to human breast carcinoma is a host myofibroblast-mediated collagenous response exhibiting synergistic effects on tumor progression. Although many paracrine interactions between breast carcinoma cells and myofibroblasts have been characterized, the event(s) which initiate desmoplasia have remained undefined. Our studies utilized c-rasH transfected MCF-7 cells which overexpress ras p2l and which are weakly tumorigenic in ovariectomized nude mice. The xenografts are desmoplastic and comprised of 30% myofibroblasts and 60 mg/g of interstitial collagen. In situ hybridization studies of these xenografts reveal a stromal gene expression pattern (stromelysin-3, IGF-II and TIMP-1) identical to that observed in human tumor desmoplasia. 17-beta estradiol increases c-rasH MCF-7 growth but abolishes desmoplasia. c-rasH MCF-7 in vitro constitutively produce myofibroblast mitogenic activity which competes with PDGF in a receptor binding assay. This myofibroblast mitogenic activity is unaltered by 17-beta estradiol/tamoxifen pretreatment in vitro. Transfection of c-rasH MCF-7 with a PDGF-A dominant negative mutant, 1308, produced by site-directed mutagenesis (serine-->cysteine129) reduces both homo- and heterodimer secretion of PDGF by as much as 90% but does not interfere with the secretion of other growth factors. Clones with low PDGF, though tumorigenic, are non-desmoplastic. Our results suggest that breast carcinoma-secreted PDGF is the major initiator of tumor desmoplasia.