Effect of cannabidiol on human gingival fibroblast extracellular matrix metabolism: MMP production and activity, and production of fibronectin and transforming growth factor ß.

BACKGROUND AND OBJECTIVE: Marijuana (Cannabis sativa) use may be associated with gingival enlargement, resembling that caused by phenytoin. Cannabidiol (CBD), a nonpsychotropic Cannabis derivative, is structurally similar to phenytoin. While there are many reports on effects of phenytoin on human gingival fibroblasts, there is no information on effects of Cannabis components on these cells. The objective of this study was to determine effects of CBD on human gingival fibroblast fibrogenic and matrix-degrading activities. MATERIAL AND METHODS: Fibroblasts were incubated with CBD in serum-free medium for 1-6 d. The effect of CBD on cell viability was determined by measuring activity of a mitochondrial enzyme. The fibrogenic molecule transforming growth factor ß and the extracellular matrix molecule fibronectin were measured by ELISA. Pro-MMP-1 and total MMP-2 were measured by ELISA. Activity of MMP-2 was determined via a colorimetric assay in which a detection enzyme is activated by active MMP-2. Data were analysed using ANOVA and Scheffe's F procedure for post hoc comparisons. RESULTS: Cannabidiol had little or no significant effect on cell viability. Low CBD concentrations increased transforming growth factor ß production by as much as 40% (p < 0.001), while higher concentrations decreased it by as much as 40% (p < 0.0001). Cannabidiol increased fibronectin production by as much as approximately 100% (p < 0.001). Lower CBD concentrations increased MMP production, but the highest concentrations decreased production of both MMPs (p < 0.05) and decreased MMP-2 activity (p < 0.02). CONCLUSION: The data suggest that the CBD may promote fibrotic gingival enlargement by increasing gingival fibroblast production of transforming growth factor ß and fibronectin, while decreasing MMP production and activity.

Tumor necrosis factor-alpha-stimulated membrane type 1-matrix metalloproteinase production is modulated by epidermal growth factor receptor signaling in human gingival fibroblasts.

BACKGROUND AND OBJECTIVES: Membrane type 1-matrix metalloproteinase (MT1-MMP) is a collagenolytic enzyme involved in connective tissue remodeling. In periodontal tissues, either cytokines or growth factors regulate the production of proteolytic enzymes. Mice deficient in epidermal growth factor receptor (EGFR) show a reduced expression of MT1-MMP, suggesting that this receptor may play an important role in MT1-MMP production. The present study evaluated the role of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and EGFR in the production of MT1-MMP in gingival fibroblasts. MATERIAL AND METHODS: Primary cultures of human gingival fibroblasts were cultured over plastic or a type I collagen matrix and stimulated with TNF-alpha and EGF. A selective EGFR inhibitor (AG1478) was used to interfere with this signaling pathway. Production of MT1-MMP and activation of proMMP-2 were studied using Western blot and gelatin zymography, respectively. Activation of EGFR signaling was assessed through immunoprecipitation and Western blot. Expression of EGFR ligands was determined through reverse transcriptase-polymerase chain reaction. RESULTS: Treatment of gingival fibroblasts cultured over a collagen matrix with TNF-alpha stimulated proMMP-2 activation and MT1-MMP production. However, after using AG1478, both responses were inhibited. Tumor necrosis factor-alpha induced EGFR transactivation and stimulated the expression of the mRNA for the EGFR ligands heparin binding-epidermal growth factor (HB-EGF) and transforming growth factor-alpha (TGF-alpha). CONCLUSIONS: The present study shows that TNF-alpha may stimulate MT1-MMP production through transactivation of EGFR. Tumor necrosis factor-alpha may also modulate the expression of the EGFR ligands TGF-alpha and HB-EGF. Production of MT1-MMP by TNF-alpha requires interaction with EGFR, suggesting that tissue remodeling is controlled by cross-communication between diverse signaling pathways in gingival fibroblasts.

Tranexamic acid can inhibit tongue squamous cell carcinoma invasion in vitro.

OBJECTIVES: Tranexamic acid (TA) is an inhibitor of plasminogen activation commonly used in surgery. Plasmin, the end product of plasminogen activation, degrades fibrin in the thrombus, leading to thrombolysis. However, plasmin is also associated with progression of several cancers and with cancer-associated matrix metalloproteinase-9 (MMP-9) activation. As the gelatinases MMP-2 and -9 are involved in cancer progression, several antigelatinolytic drugs have been developed as potential anticancer therapeutics. We previously developed gelatinases targeting peptide CTT1 capable of inhibiting carcinoma growth. STUDY DESIGN: The effects of TA and CTT1 on tongue carcinoma aggressiveness were evaluated in an in vitro assay of human HSC-3 and SCC-25 cells. MATERIALS AND METHODS: The cells were cultured with or without TA and CTT1 and their proMMP-9 production and activation were analysed with Western immunoblotting and gelatin zymography. Their effects on tongue carcinoma invasion were analysed in a Matrigel assay. RESULTS: Tranexamic acid alone and in combination with CTT1 can inhibit tongue SCC invasion in vitro, at least partially explained by its property of reducing the plasmin-mediated activation of proMMP-9. CONCLUSIONS: These data suggest that patients undergoing surgical therapy for large oral malignancies may cobenefit from prolonged TA therapy, because of its antithrombolytic and antitumour properties.

Matrix metalloproteinase-2 and -9 are induced differently by metal nanoparticles in human monocytes: The role of oxidative stress and protein tyrosine kinase activation.

Recently, many studies have shown that nanoparticles can translocate from the lungs to the circulatory system. As a particulate foreign body, nanoparticles could induce host responses such as reactive oxygen species (ROS) generation, inflammatory cytokine and matrix metalloproteinase (MMP) release which play a major role in tissue destruction and remodeling. However, the direct effects of nanoparticles on leukocytes, especially monocytes, are still unclear. The objective of the present study was to compare the ability of Nano-Co and Nano-TiO(2) to cause alteration of transcription and activity of MMPs and to explore possible mechanisms. We hypothesized that non-toxic doses of some transition metal nanoparticles stimulate an imbalance of MMP/TIMP that cause MMP production that may contribute to their health effects. To test this hypothesis, U937 cells were treated with Nano-Co and Nano-TiO(2) and cytotoxic effects and ROS generation were measured. The alteration of MMP-2 and MMP-9 expression and activity of MMP-2 and MMP-9 after exposure to these metal nanoparticles were subsequently determined. To investigate the potential signaling pathways involved in the Nano-Co-induced MMP activation, the ROS scavengers or inhibitors, AP-1 inhibitor, and protein tyrosine kinase (PTK) inhibitors were also used to pre-treat U937 cells. Our results demonstrated that exposure of U937 cells to Nano-Co, but not to Nano-TiO(2), at a dose that does not cause cytotoxicity, resulted in ROS generation and up-regulation of MMP-2 and MMP-9 mRNA expression(..) Our results also showed dose- and time-related increases in pro-MMP-2 and pro-MMP-9 gelatinolytic activities in conditioned media after exposure of U937 cells to Nano-Co, but not to Nano-TiO(2). Nano-Co-induced pro-MMP-2 and pro-MMP-9 activity increases were inhibited by pre-treatment with ROS scavengers or inhibitors. We also demonstrated dose- and time-related decreases in tissue inhibitors of metalloproteinases 2 (TIMP-2) in U937 cells after exposure to Nano-Co, but not to Nano-TiO(2). However, neither Nano-Co nor Nano-TiO(2) exposure led to any transcriptional change of TIMP-1. The decrease of TIMP-2 after exposure to Nano-Co was also inhibited by pre-treatment with ROS scavengers or inhibitors. Our results also showed that pre-treatment of U937 cells with AP-1 inhibitor, curcumin, or the PTK specific inhibitor, herbimycin A or genistein, prior to exposure to Nano-Co, significantly abolished Nano-Co-induced pro-MMP-2 and-9 activity. Our results suggest that Nano-Co causes an imbalance between the expression and activity of MMPs and their inhibitors which is mediated by the AP-1 and tyrosine kinase pathways due to oxidative stress.

Lercanidipine reduces matrix metalloproteinase-2 activity and reverses vascular dysfunction in renovascular hypertensive rats.

Increased expression/activity of matrix metalloproteinases (MMPs), especially MMP-2, plays a role in the vascular alterations induced by hypertension, and increased oxidative stress is a major factor activating MMPs. Here, we hypothesized that lercanidipine, a calcium channel blocker, could attenuate the increases in oxidative stress and MMP-2 expression/activity in the two-kidney, one-clip (2K-1C) hypertensive rats. Sham-operated or 2K-1C hypertension rats were treated with lercanidipine 2.5 mg/kg/day (or vehicle) starting three weeks after hypertension was induced. Systolic blood pressure was monitored weekly. After five weeks of treatment, aortic rings were isolated to assess endothelium-dependent and independent relaxations. Quantitative morphometry of structural changes in the aortic wall were studied in hematoxylin/eosin sections. Aortic MMP-2 levels were determined by gelatin zymography. Aortic MMP-2/tissue inhibitor of metalloproteinases (TIMP)-2 mRNA levels were determined by quantitative real-time RT-PCR. Plasma thiobarbituric acid reactive substances concentrations were determined using a fluorometric method. Lercanidipine attenuated 2K-1C hypertension (224+/-12 versus 183+/-11 mm Hg in 2K-1C rats and 2K-1C + Lercandipine rats, respectively; P<0.01) and prevented the reduction in endothelium-dependent vasorelaxation found in 2K-1C rats. Increased MMP-2 and Pro-MMP-2 levels were found in the aortas of 2K-1C rats (all P<0.05). Lercandipine attenuated 2K-1C-induced increases in MMP-2 by more than 60% and blunted 2K-1C-induced increases in oxidative stress (both P<0.001). While hypertension-induced significant aortic wall hypertrophy and approximately 9-fold increases in the ratio of MMP-2/TIMP-2 mRNA expression (both P<0.05), lercandipine did not affect these changes. These results suggest that lercanidipine produces antihypertensive effects and reverses the endothelial dysfunction associated with 2K-1C hypertension, probably through mechanisms involving antioxidant effects leading to lower MMP-2 activation.

Lercanidipine decreases vascular matrix metalloproteinase-2 activity and protects against vascular dysfunction in diabetic rats.

Abnormal matrix metalloproteinases (MMPs) activity causes cardiovascular diseases. Because hyperglycemia increase MMPs activities through increased oxidative stress, we hypothesized that antioxidant effects produced by lercanidipine could attenuate the increases in MMP-2 expression/activity in diabetic rats. Control and diabetic (alloxan-induced diabetes) rats received lercanidipine 2.5 mg/kg/day (or tap water) starting three weeks after alloxan (or vehicle) injections. Blood pressure was monitored weekly. After six weeks of treatment, vascular reactivity and structural changes were assessed in aortic rings. MMP-2 levels were determined by gelatin zymography, and MMP-2/tissue inhibitor of metalloproteinases (TIMP)-2 mRNA levels were determined by quantitative real time RT-PCR. Plasma thiobarbituric acid reactive substances concentrations were determined by fluorimetry. Lercanidipine produced antihypertensive effects (201+/-5 vs. 163+/-7 mm Hg in diabetic rats untreated and treated with lercanidipine, respectively; P<0.01) and reversed the impairment in endothelium-dependent vasorelaxation in diabetic rats. Increased MMP-2 and Pro-MMP-2 levels were found in the aortas of diabetic rats (both P<0.001). Lercandipine attenuated the increases in oxidative stress and in MMP-2 (both P<0.05). While diabetes induced no major structural changes, it caused a 16-fold increase in the ratio of MMP-2/TIMP-2 mRNA expression, which was completely reversed by lercanidipine (both P<0.001). These results show that antioxidant and beneficial vascular effects produced by lercanidipine in diabetic rats are associated with reversion of the imbalance in vascular MMP-2/TIMP-2 expression.

Imatinib inhibits colorectal cancer cell growth and suppresses stromal-induced growth stimulation, MT1-MMP expression and pro-MMP2 activation.

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.

Local and systemic pathophysiological alterations induced by a serine proteinase from the venom of the snake Bothrops jararacussu.

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.

Effector-induced Syk-mediated phosphorylation in human erythrocytes.

Band 3 (AE1), the most prominent polypeptide of the human erythrocyte membrane, becomes heavily tyrosine phosphorylated following treatment of intact cells with protein tyrosine phosphatase inhibitors such as diamide, pervanadate, vanadate, or N-ethylmaleimide (NEM). The mechanism underlying this tyrosine phosphorylation is thought to involve the sequential action of two protein tyrosine kinases, Syk (p72syk) and Lyn (p53/56lyn). While Lyn catalysed phosphorylation appears to be strictly dependent on prior phosphorylation of Tyr8 and 21 of band 3 by Syk, little is known about the mechanism of induction of Syk phosphorylation. Data presented here show that both the fraction of Syk that associates with the membrane and the extent of phosphorylation of band 3 differ in response to the above inhibitors. While diamide and NEM stimulate syk translocation to the membrane during their induction of band 3 tyrosine phosphorylation, pervanadate and vanadate induce no change in kinase distribution. Moreover, diamide and NEM-induced Syk recruitment to the membrane are phosphotyrosine independent and involve their preferential association with Triton X-100-insoluble membrane skeletons. Together these data reveal a complex process controlling the association and catalytic activity of protein tyrosine kinases syk and lyn with the human erythrocyte membrane.

Hydrogen peroxide-mediated downregulation of matrix metalloprotease-2 in indomethacin-induced acute gastric ulceration is blocked by melatonin and other antioxidants.

Gastric mucosal damage is directly associated with extracellular matrix degradation in which matrix metalloproteinases (MMPs) play a crucial role. Remodeling of connective tissues and loss of tissue integrity due to the action of MMPs are reported in several inflammatory diseases, including gastric ulcer. Indomethacin-induced gastric ulceration involves the generation of reactive oxygen species (ROS) and a reduction in MMP-2 transcription and translation. Our aim was to identify the mechanism for suppression of MMP-2 activity by ROS during acute ulceration and further to examine the possible actions of antioxidants, especially melatonin, during healing. Melatonin (N-acetyl-5-methoxytryptamine) blocked hydroxyl radical and nitrite anion generation, protein oxidation, mucosal cell disruption, and MMP-2 downregulation. In addition, suppression of MMP-2 activity by H2O2 in a dose- and time-dependent manner in vitro is blocked by melatonin, omeprazole, and curcumin. We observed that melatonin and other antioxidants (e.g., curcumin and omeprazole) offered gastroprotection in vivo by upregulation of suppressed MMP-2 expression and activity at the level of secretion and synthesis. Moreover, antioxidants reversed the suppression of MMP-2 expression by upregulation of MT1-MMP and downregulation of TIMP-2. Hence, we hypothesize that antioxidants exerted protection against H2O2-mediated inactivation and downregulation of MMP-2 expression during onset of indomethacin-induced ulceration.

Indanocine, a microtubule-binding indanone and a selective inducer of apoptosis in multidrug-resistant cancer cells.

BACKGROUND: Certain antimitotic drugs have antitumor activities that apparently result from interactions with nontubulin components involved in cell growth and/or apoptotic cell death. Indanocine is a synthetic indanone that has been identified by the National Cancer Institute's Developmental Therapeutics Program as having antiproliferative activity. In this study, we characterized the activity of this new antimitotic drug toward malignant cells. METHODS: We tested antiproliferative activity with an MTT [i.e., 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay, mitochondrial damage and cell cycle perturbations with flow cytometry, caspase-3 activation with fluorometry, alterations of the cytoskeletal components with immunofluorescence, and antimicrotubule activity with a tubulin polymerization assay. RESULTS/CONCLUSIONS: Indanocine is a cytostatic and cytotoxic indanone that blocks tubulin polymerization but, unlike other antimitotic agents, induces apoptotic cell death in stationary-phase multidrug-resistant cancer cells at concentrations that do not impair the viability of normal nonproliferating cells. Of the seven multidrug-resistant cell lines tested, three (i.e., MCF-7/ADR, MES-SA/DX5, and HL-60/ADR) were more sensitive to growth inhibition by indanocine than were their corresponding parental cells. Confluent multidrug-resistant cells (MCF-7/ADR), but not drug-sensitive cancer cells (MCF-7) or normal peripheral blood lymphocytes, underwent apoptotic cell death 8-24 hours after exposure to indanocine, as measured by sequential changes in mitochondrial membrane potential, caspase activity, and DNA fragmentation. Indanocine interacts with tubulin at the colchicine-binding site, potently inhibits tubulin polymerization in vitro, and disrupts the mitotic apparatus in dividing cells. IMPLICATIONS: The sensitivity of stationary multidrug-resistant cancer cells to indanocine suggests that indanocine and related indanones be considered as lead compounds for the development of chemotherapeutic strategies for drug-resistant malignancies.

Hsp27 functions as a negative regulator of cytochrome c-dependent activation of procaspase-3.

The release of mitochondrial cytochrome c by genotoxic stress induces the formation of a cytosolic complex with Apaf-1 (mammalian CED4 homolog) and thereby the activation of procaspase-3 (cas-3) and procaspase-9 (cas-9). Here we demonstrate that heat-shock protein 27 (Hsp27) inhibits cytochrome c (cyt c)-dependent activation of cas-3. Hsp27 had no effect on cyt c release, Apaf-1 and cas-9 activation. By contrast, our results show that Hsp27 associates with cas-3, but not Apaf-1 or cas-9, and inhibits activation of cas-3 by cas-9-mediated proteolysis. Furthermore, the present results demonstrate that immunodepletion of Hsp27 depletes cas-3. Importantly, treatment of cells with DNA damaging agents dissociates the Hsp27/cas-3 complex and relieves inhibition of cas-3 activation. These findings define a novel function for Hsp27 and provide the first evidence that a heat shock protein represses cas-3 activation.

Release of cytochrome c and activation of pro-caspase-9 following lysosomal photodamage involves Bid cleavage.

Photodynamic therapy (PDT) protocols employing lysosomal sensitizers induce apoptosis via a mechanism that causes cytochrome c release prior to loss of mitochondrial membrane potential (DeltaPsi(m)). The current study was designed to determine how lysosomal photodamage initiates mitochondrial-mediated apoptosis in murine hepatoma 1c1c7 cells. Fluorescence microscopy demonstrated that the photosensitizer N-aspartyl chlorin e6 (NPe6) localized to the lysosomes. Irradiation of cultures preloaded with NPe6 induced the rapid destruction of lysosomes, and subsequent cleavage/activation of Bid, pro-caspases-9 and -3. Pro-caspase-8 was not activated. Release of cytochrome c occurred at about the time of Bid cleavage and preceded the loss of DeltaPsi(m). Extracts of purified lysosomes catalyzed the in vitro cleavage of cytosolic Bid, but not pro-caspase-3 activation. Pharmacological inhibition of cathepsin B, L and D activities did not suppress Bid cleavage or pro-caspases-9 and -3 activation. These studies demonstrate that photodamaged lysosomes trigger the mitochondrial apoptotic pathway by releasing proteases that activate Bid.

Apoptotic pathway activation from mitochondria and death receptors without caspase-3 cleavage in failing human myocardium: fragile balance of myocyte survival?

OBJECTIVES: Activation of the caspase cascade through the mitochondrial and/or death receptor pathway was investigated in the failing human myocardium, in which the mode and extent of the cascade activation are unknown. BACKGROUND: In terminal heart failure, a loss of cardiomyocytes by overload-induced apoptosis is an attractive mechanism, explaining the progressive character of the disease. However, its relevance is unclear, because the specificity of probes for apoptotic deoxyribonucleic acid damage is under debate. METHODS: Left ventricular specimens from 36 explanted failing and 21 nonfailing donor hearts were used for messenger ribonucleic acid detection by semiquantitative reverse-transcription polymerase chain reaction. From these groups, immunoblot analysis was performed in samples from nine failing and six nonfailing donor hearts. RESULTS: In terminally failing hearts, there was a significant accumulation of cytochrome c in the cytosol, which was associated with activation of caspase-9 and downregulation of its inhibitor, caspase-9S. Similarly, the death receptor-induced pathway revealed activation of caspase-8, combined with downregulation of its inhibitors, flice-like inhibitory protein-L (FLIP(L)) and FLIP(S). The unspecific caspase inhibitors, XIAP, hIAP-1 and hIAP-2, were also downregulated. However, the terminal effector caspase-3 was not activated, and its substrate gelsolin, acting in its uncleaved form as a feedback inhibitor of caspase-3, was not cleaved. CONCLUSIONS: In the terminally failing human myocardium, the caspase cascade is partially activated in the presence of a consistent phenotype shift toward enhanced susceptibility to apoptosis. Although the system is still under a fragile control, the partial initiation of the apoptotic program may be of functional relevance also for the surviving cardiomyocytes.

N-terminal domain of the dual-targeted pea glutathione reductase signal peptide controls organellar targeting efficiency.

Import of nuclear-encoded proteins into mitochondria and chloroplasts is generally organelle specific and its specificity depends on the N-terminal signal peptide. Yet, a group of proteins known as dual-targeted proteins have a targeting peptide capable of leading the mature protein to both organelles. We have investigated the domain structure of the dual-targeted pea glutathione reductase (GR) signal peptide by using N-terminal truncations. A mutant of the GR precursor (pGR) starting with the second methionine residue of the targeting peptide, pGRdelta2-4, directed import into both organelles, negating the possibility that dual import was controlled by the nature of the N terminus. The deletion of the 30 N-terminal residues (pGRdelta2-30) inhibited import efficiency into chloroplasts substantially and almost completely into mitochondria, whereas the removal of only 16 N-terminal amino acid residues (pGRdelta2-16) resulted in the strongly stimulated mitochondrial import without significantly affecting chloroplast import. Furthermore, N-terminal truncations of the signal peptide (pGRdelta2-16 and pGRdelta2-30) greatly stimulated the mitochondrial processing activity measured with the isolated processing peptidase. These results suggest a domain structure for the dual-targeting peptide of pGR and the existence of domains controlling organellar import efficiency therein.

A new approach to treat tissue destruction in periodontitis with chemically modified dextran polymers.

Periodontitis are diseases of the supportive tissues of the teeth provoked by bacteria and characterized by gingival inflammation and bone destruction. We have developed a new strategy to repair tissues by administrating agents (RGTA) that mimic heparan sulfates by protecting selectively some of the growth factors naturally present within the injured tissue and interfering with inflammation. After periodontitis induction in hamsters, the animals were left untreated or received weekly i.m. injections of RGTA1507 at a dose of 100 microg/kg, 400 microg/kg, 1.5 mg/kg, or 15 mg/kg for 4 wk. RGTA treatment significantly reduced gingival tissue inflammation, thickened the pocket epithelium by increasing cell proliferation, and enhanced collagen accumulation in the gingiva. A marked reduction in bone loss was observed, resulting from depression of osteoclasia and robust stimulation of bone formation at the dose of 1.5 mg/kg. RGTA treatment for 8 wk at this dose reversed macroscopic bone loss, sharply contrasting with the extensive bone destruction in the untreated animals. RGTA treatment decreased gelatinase A (MMP-2) and B (MMP-9) pro-forms in gingival tissues. Our data indicate that a 4 wk treatment dose-dependently attenuated gingival and bone manifestations of the disease, whereas a longer treatment restored alveolar bone close to controls. By modulating and coordinating host responses, RGTA has unique therapeutic properties and is a promising candidate for the treatment of human periodontitis.

Platelet-activating factor (PAF) induces activation of matrix metalloproteinase 2 activity and vascular endothelial cell invasion and migration.

Tumor-induced angiogenic responses lead to complex phenotypic changes in vascular endothelial cells, which must coordinate the expression of both proteases and protease inhibitors prior to the proliferation and invasion of surrounding stroma. Matrix metalloproteinase 2 (MMP2), which degrades Type IV collagen, is produced as proMMP2. proMMP2 is activated in part through its interactions with membrane Type 1 MMP (MT1-MMP) and tissue inhibitor of matrix metalloproteinase 2 (TIMP2). In this study, we demonstrate that platelet-activating factor (PAF) is a potent inducer of human umbilical vein endothelial cell (HUVEC) migration and invasion, which is attenuated by PAF receptor antagonists, and that PAF receptor antagonists inhibit the migration and invasion of HUVEC mediated by medium conditioned by a prostatic carcinoma cell line. We confirm that PAF receptor antagonists inhibit proliferation of HUVEC grown in rich growth medium. We show that PAF increases mRNA levels for MT1-MMP and TIMP2, followed by increased temporal conversion of latent proMMP2 to MMP2. Finally, we demonstrate that the ratio of MT1-MMP to TIMP2 in membrane preparations from PAF-stimulated HUVEC is 1.6:1, approximating the hypothesized ideal ratio of 2:1 necessary for the conversion of proMMP2 to MMP2. Our data support the involvement of PAF in vascular endothelial cell migration and invasion.

Relationship of the plasminogen activator/plasmin cascade to osteoclast invasion and mineral resorption in explanted fetal metatarsal bones.

An attempt was made to establish whether the activation of plasminogen into plasmin is necessary either for the preparatory phases to bone resorption, involving the recruitment of osteoclast precursors, their migration toward mineralized surfaces, and their final differentiation, or for the subsequent osteoclastic resorption phase. 45Ca-labeled fetal (17 day) mouse metatarsals were cultured under conditions in which they pursue their modeling for a few days. In this model, the resorption phase, monitored by the release of 45Ca into the medium, is entirely dependent on the preparatory phases affecting osteoclast precursors. It was, as expected, stimulated by parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 and inhibited by calcitonin. PTH also enhanced the activity of tissue-type plasminogen activator (PA) in extracts of metatarsals but not that of urokinase (which is, however, the main PA present in the mouse fetal metatarsal culture model). The resorption processes were not dependent on the presence of plasminogen in the media, even when the rudiments were precultured with tranexamic acid to remove their endogenous plasminogen. Moreover, they were not influenced by inhibitors of plasmin, either the plasma inhibitors alpha 2-antiplasmin, alpha 2-macroglobulin, and alpha 1-antitrypsin, or aprotinin, which was tested under a variety of conditions. Aprotinin also did not influence the resorption (loss of calcium and hydroxyproline) of 19 day fetal mouse calvariae cultured with PTH in a medium devoid of plasminogen. It is concluded that the various steps implicated in the bone resorption processes that occur in the metatarsals and in the calvariae culture models are not dependent on the activity of plasmin. The function of PAs in bone, however, could be exerted through direct proteolysis of extracellular proteins other than plasminogen or be mediated by a molecular structural domain distinct from their catalytic domain.

Linear mixed irreversible inhibition of the autocatalytic activation of zymogens. Kinetic analysis checked by simulated progress curves.

Autocatalytic zymogen activation is a phenomenon of great importance for understanding some fundamental physiological processes involved in the enzyme regulation of gastrointestinal-tract enzymes, blood coagulation, fibrinolysis and the complement system. Examples of such processes are the activation of prekallikrein, trypsinogen and pepsinogen, all of which are controlled by natural proteinase inhibitors. This work studies the kinetics of a general autocatalytic zymogen activation process overlapped by two two-step irreversible inhibitions, i.e. a linear mixed irreversible inhibition. The kinetic equations for the whole course of the reaction are derived for this mechanism. In addition, we determine the corresponding kinetics for a number of particular cases of the general model analyzed, i.e. for reversible and irreversible non-competitive, competitive and uncompetitive inhibition systems which are considered particular cases of the general mechanism studied. The kinetic behavior of the system is related to a parameter, a dimensionless quantity, which shows whether the inhibition or the activation route prevails, in a similar way to that which we have previously carried out for other mechanisms. Finally, based on the kinetic equations obtained, a procedure for discriminating between the different mechanisms considered is suggested. The results of this contribution can be directly applied to most physiological autocatalytic zymogen activations in the presence of an inhibitor, allowing their complete kinetic characterization and suggesting procedures for varying the relative weight of the catalytic and inhibition routes or for changing the predominant route.

Human progelatinase A can be activated by matrilysin.

The activation of human progelatinase A by other matrix metalloproteinases was studied by following both the loss of its N-terminal propeptide and the accompanying increase in the rate of hydrolysis of a synthetic substrate. Activated stromelysin 1 was unable to cause any activation of progelatinase A beyond that slowly occurring by autolysis, but an 8 h incubation with activated matrilysin was able to produce 64% of the activity generated by incubation with (4-aminophenylmercuric)acetate (APMA). Wild-type progelatinase A and a mutant proenzyme that cannot become active were both cleaved by matrilysin to a lower molecular weight species that had lost the propeptide. This shows that matrilysin activates progelatinase A by removing the propeptide in a process that does not require any autolytic cleavages.