Matrix Metalloproteinase-2 (MMP-2) Gene Deletion Enhances MMP-9 Activity,Impairs PARP-1 Degradation,and Exacerbates Hepatic Ischemia and Reperfusion Injury in Mice

Hepatic ischemia and reperfusion injury (IRI) is an inflammatory condition and a significant cause of morbidity and mortality after surgery. Matrix metalloproteinases (MMPs) have been widely implicated in the pathogenesis of inflammatory diseases. Among the different MMPs, gelatinases (MMP-2 and MMP-9) are within the most prominent MMPs detected during liver IRI. While the role of MMP-9 in liver damage has been fairly documented, direct evidence of the role for MMP-2 activity in hepatic IRI remains to be established. Due to the lack of suitable inhibitors to target individual MMPs in vivo, gene manipulation is as an essential tool to assess MMP direct contribution to liver injury. Hence, we used MMP-2^-/- deficient mice and MMP-2^+/+ wild-type littermates to examine the function of MMP-2 activity in hepatic IRI. MMP-2 expression was detected along the sinusoids of wild-type livers before and after surgery and in a small population of leukocytes post-IRI. Compared to MMP-2^+/+ mice, MMP-2 null (MMP-2^-/-) mice showed exacerbated liver damage at 6, 24, and 48 hours post-reperfusion, which was fatal in some cases. MMP-2 deficiency resulted in upregulation of MMP-9 activity, spontaneous leukocyte infiltration in naïve livers, and amplified MMP-9-dependent transmigration of leukocytes in vitro and after hepatic IRI. Moreover, complete loss of MMP-2 activity impaired the degradation of poly (ADP-ribose) polymerase (PARP-1) in extensively damaged livers post-reperfusion. However, the administration of a PARP-1 inhibitor to MMP-2 null mice restored liver preservation to almost comparable levels of MMP-2^+/+ mice post-IRI. Deficient PARP-1 degradation in MMP-2-null sinusoidal endothelial cells correlated with their increased cytotoxicity, evaluated by the measurement of LDH efflux in the medium. In conclusion, our results show for the first time that MMP-2 gene deletion exacerbates liver IRI. Moreover, they offer new insights into the MMP-2 modulation of inflammatory responses, which could be relevant for the design of new pharmacological MMP-targeted agents to treat hepatic IRI.     

Investigation of the budding process in Tethya citrina and Tethya aurantium (Porifera, Demospongiae)

The budding process has been studied in two congeneric Mediterranean species belonging to Tethya from different sampling sites: Marsala and Venice Lagoons (Tethya citrina); Marsala Lagoon and Porto Cesareo Basin (Tethya aurantium). Buds, connected to the adult by a spiculated stalk, differ between the two species in morphology and size, since those of T. citrina are small with elongated bodies, showing only a few spicules protruding from the apical region, whereas those of T. aurantium are round, larger, and show spicules radiating from the peripheral border. In T. citrina, cells with inclusions, varying in electron density and size, represent the main cell types of the buds. In T. aurantium, the cell component shows a major diversification, resulting from spherulous cells, grey cells, vacuolar cells and peculiar micro-vesicle cells. Neither canals nor choanocyte chambers were observed in the buds of the two species. In T. citrina, bud production is similar in both sampling sites. In T. aurantium, budding occurs more rarely in Porto Cesareo Basin, probably in relation with environmental factors, such as the covering of the cortex by sediment and micro-algae. Finally, in the buds of both species, the spicule size does not differ from that of the cortex of the adult sponges, further supporting the main involvement of the cortex in organizing the skeletal architecture of the buds.     

Interspecific variation in arrangement and morphology of micrasters of Tethya species (Porifera,Demospongiae)

Summary A new distinctive feature between the two Mediterranean species of Tethya, T. aurantium and T. citrina has been found in the body arrangement of different types of micrasters. Contrary to the previous assumptions, T. aurantium has two clearly distinct categories of micrasters: the chiaster-tylaster in the cortex and the larger, slender oxyaster in the choanosome. T. citrina has only slightly differentiated micraster sets in the cortex and choanosome; in the latter the shape of micrasters is close to that of oxyasters. SEM analysis shows that differences in micraster shape depend on the cylindrical or conical form of rays and on the distribution, density and strength of the microspines along their axis. The relationship between the degree of micraster differentiation and the development of the cortex in the two species is discussed.     

The Inhibitory Effect of C-phycocyanin Containing Protein Extract (C-PC Extract) on Human Matrix Metalloproteinases (MMP-2 and MMP-9) in Hepatocellular Cancer Cell Line (HepG2)

Spirulina platensis :have been studied for several biological activities. In the current study C-phycocyanin containing protein extract (C-PC extract) of Spirulina platensis have been studied for its effect on human matrix metalloproteinases (MMP-1, MMP-2 and MMP-9) and tissue inhibitors of MMPs (TIMP-1 and TIMP-2). In the present study, breast cancer cell line (MDA-MB 231) and hepatocellular cancer cell line (HepG2) were examined for inhibition of MMPs at different levels of expression after C-PC extract treatment. Herein, we have demonstrated that C-PC extract significantly reduced activity of MMP-2 by 55.13% and MMP-9 by 57.9% in HepG2 cells at 15 μg concentration. Additionally, the treatment has reduced mRNA expression of MMP-2 and MMP-9 at 20 μg concentration by 1.65-folds and 1.66-folds respectively. The C-PC extract treatment have also downregulated a mRNA expression of TIMP-2 by 1.12 folds at 20 μg concentration in HepG2 cells. Together, these results indicate that C-PC, extract successfully inhibited MMP-2 and -9 at different levels of expression and TIMP-2 at a mRNA expression level; however, extract did not have any effect on MMP-1 expressed in MDA-MB231 and TIMP-1 expressed in HepG2 cells as well as the exact mechanism of inhibition of MMP-2, MMP-9 and TIMP-2 remained unclear.     

Contributions of Ocular Surface Components to Matrix-Metalloproteinases (MMP)-2 and MMP-9 in Feline Tears following Corneal Epithelial Wounding

Purpose

This study investigated ocular surface components that contribute to matrix-metalloproteinase (MMP)-2 and MMP-9 found in tears following corneal epithelial wounding.

Methods

Laboratory short-haired cats underwent corneal epithelial debridement in one randomly chosen eye (n = 18). Eye-flush tears were collected at baseline and during various healing stages. Procedural control eyes (identical experimental protocol as wounded eyes except for wounding, n = 5) served as controls for tear analysis. MMP activity was analyzed in tears using gelatin zymography. MMP staining patterns were evaluated in ocular tissues using immunohistochemistry and used to determine MMP expression sites responsible for tear-derived MMPs.

Results

The proMMP-2 and proMMP-9 activity in tears was highest in wounded and procedural control eyes during epithelial migration (8 to 36 hours post-wounding). Wounded eyes showed significantly higher proMMP-9 in tears only during and after epithelial restratification (day 3 to 4 and day 7 to 28 post-wounding, respectively) as compared to procedural controls (p<0.05). Tears from wounded and procedural control eyes showed no statistical differences for pro-MMP-2 and MMP-9 (p>0.05). Immunohistochemistry showed increased MMP-2 and MMP-9 expression in the cornea during epithelial migration and wound closure. The conjunctival epithelium exhibited highest levels of both MMPs during wound closure, while MMP-9 expression was reduced in conjunctival goblet cells during corneal epithelial migration followed by complete absence of the cells during wound closure. The immunostaining for both MMPs was elevated in the lacrimal gland during corneal healing, with little/no change in the meibomian glands. Conjunctival-associated lymphoid tissue (CALT) showed weak MMP-2 and intense MMP-9 staining.

Conclusions

Following wounding, migrating corneal epithelium contributed little to the observed MMP levels in tears. The major sources assessed in the present study for tear-derived MMP-2 and MMP-9 following corneal wounding are the lacrimal gland and CALT. Other sources included stromal keratocytes and conjunctiva with goblet cells.     

PAC-1 and isatin derivatives are weak matrix metalloproteinase inhibitors

Background

Dysregulation of apoptotic cell death is observed in a large number of pathological conditions. As caspases are central enzymes in the regulation of apoptosis, a large number of procaspase-activating compounds (PAC-1 derivatives) and inhibitors (isatin derivatives) have been developed. Matrix metalloproteinases (MMPs) have been shown to have a dual role in apoptosis. Hence compounds that either activate or inhibit caspases should ideally not affect MMPs. As many PAC-1 derivatives contain a zinc chelating ortho-hydroxy N-acyl hydrazone moiety and isatin derivatives has two carbonyl groups on the indole core, it was of interest to determine to which extent these compounds can inhibit MMPs.

Methods

Eight PAC-1 and five isatin derivatives were docked into MMP-9 and MMP-14. The same compounds were synthesized, characterized, purified and tested as inhibitors of MMP-9 and MMP-14, using fluorescence quenched peptide and biological substrates. Some of the compounds were also tested for fluorescence quenching.

Results

Molecular docking suggested that the different compounds can bind to the MMP active sites. However, kinetic studies showed that neither of these compounds was a strong MMP inhibitor. IC₅₀ values over 100 μM were obtained after the enzyme activities were corrected for quenching. These IC₅₀ values are far above the concentrations needed to activate or inhibit the caspases.

Conclusion

The use of PAC-1 and isatin derivatives against caspases should have little or no effect on the activity of MMPs.

General significance

Activators and inhibitors of caspases are important potential therapeutic agents for several diseases such as cancer, diabetes and neurodegenerative disorders.     

Molecular Design of a Highly Selective and Strong Protein Inhibitor against Matrix Metalloproteinase-2 (MMP-2)

Synthetic inhibitors of matrix metalloproteinases (MMPs), designed previously, as well as tissue inhibitors of metalloproteinases (TIMPs) lack enzyme selectivity, which has been a major obstacle for developing inhibitors into safe and effective MMP-targeted drugs. Here we designed a fusion protein named APP-IP-TIMP-2, in which the ten amino acid residue sequence of APP-derived MMP-2 selective inhibitory peptide (APP-IP) is added to the N terminus of TIMP-2. The APP-IP and TIMP-2 regions of the fusion protein are designed to interact with the active site and the hemopexin-like domain of MMP-2, respectively. The reactive site of the TIMP-2 region, which has broad specificity against MMPs, is blocked by the APP-IP adduct. The recombinant APP-IP-TIMP-2 showed strong inhibitory activity toward MMP-2 (K_i^app = 0.68 pm), whereas its inhibitory activity toward MMP-1, MMP-3, MMP-7, MMP-8, MMP-9, or MT1-MMP was six orders of magnitude or more weaker (IC₅₀ > 1 μm). The fusion protein inhibited the activation of pro-MMP-2 in the concanavalin A-stimulated HT1080 cells, degradation of type IV collagen by the cells, and the migration of stimulated cells. Compared with the decapeptide APP-IP (t_½ = 30 min), APP-IP-TIMP-2 (t_½ ≫ 96 h) showed a much longer half-life in cultured tumor cells. Therefore, the fusion protein may be a useful tool to evaluate contributions of proteolytic activity of MMP-2 in various pathophysiological processes. It may also be developed as an effective anti-tumor drug with restricted side effects.     

Delineation of crosstalk between HSP27 and MMP-2/MMP-9: A synergistic therapeutic avenue for glioblastoma management

BackgroundEpithelial to mesenchymal transition (EMT) and extracellular matrix (ECM) remodeling, are the two elemental processes promoting glioblastoma (GBM). In the present work we propose a mechanistic modelling of GBM and in process establish a hypothesis elucidating critical crosstalk between heat shock proteins (HSPs) and matrix metalloproteinases (MMPs) with synergistic upregulation of EMT-like process and ECM remodeling.MethodsThe interaction and the precise binding site between the HSP and MMP proteins was assayed computationally, in-vitro and in GBM clinical samples.ResultsA positive crosstalk of HSP27 with MMP-2 and MMP-9 was established in both GBM patient tissues and cell-lines. This association was found to be of prime significance for ECM remodeling and promotion of EMT-like characteristics. In-silico predictions revealed 3 plausible interaction sites of HSP27 interacting with MMP-2 and MMP-9. Site-directed mutagenesis followed by in-vitro immunoprecipitation assay (IP) with 3 mutated recombinant HSP27, confirmed an interface stretch containing residues 29–40 of HSP27 to be a common interaction site for both MMP-2 and MMP-9. This was further validated with in-vitro IP of truncated (sans AA 29–40) recombinant HSP27 with MMP-2 and MMP-9.ConclusionThe association of HSP27 with MMP-2 and MMP-9 proteins along with the identified interacting stretch has the potential to contribute towards drug development to inhibit GBM infiltration and migration.General significanceCurrent findings provide a novel therapeutic target for GBM opening a new horizon in the field of GBM management.     

Unveiling antimicrobial activity of microalgae Chlorella sorokiniana (UKM2), Chlorella sp. (UKM8) and Scenedesmus sp. (UKM9)

Microalgae represent promising sources of bioactive compounds for pharmaceutical and industrial applications. The emergence of antibiotic resistant bacteria leads to the need to explore new cost-effective, safe, and potent bioactive compounds from the microalgae. This study aimed to investigate the potential of local microalgae for their antimicrobial properties and bioactive compounds. Three local microalgae namely Chlorella sorokiniana (UKM2), Chlorella sp. UKM8, and Scenedesmus sp. UKM9 biomass methanol extracts (ME) were prepared and tested against Gram-positive and Gram-negative bacteria. Chlorella sp. UKM8-ME showed the highest antibacterial activity. UKM8-ME minimum inhibitory concentrations were in the range of 0.312 to 6.25 mg/mL. Cytotoxicity evaluation using MTT assay showed that the microalgae methanolic extracts did not exhibit cytotoxicity against Vero-cells. The UKM8-ME was mainly containing 28 compounds from the Gas Chromatography-Mass Spectrometry (GC–MS) analysis. Major compounds of UKM8-ME included phenol (18.5%), hexadecanoic acid (18.25%), phytol (14.43%), 9,12-octadecadienoic acid (13.69%), and bicyclo[3.1.1]heptane (7.23%), which have been previously described to possess antimicrobial activity. Hence, Chlorella sp. (UKM8) methanol extracts showed promising antibacterial activity. More comprehensive studies are required to purify these antimicrobial compounds and develop our understanding on their mechanism in UKM8-ME to unleash their specific potential.     

Solvent-partitioned fractions from <Emphasis Type="Italic">Ishige okamurae</Emphasis> extract inhibit MMP-2 and MMP-9 activities in human fibrosarcoma cells in vitro

Matrix metalloproteinases (MMPs) are endopeptidases that take significant roles in extracellular matrix degradation and therefore linked with several complications such as metastasis of cancer progression, oxidative stress, and hepatic fibrosis. Ishige okamurae, a brown alga, has been reported to possess various bioactivities including but not limited to antiviral, antimicrobial, and anti-inflammatory. In this study, the potential of I. okamurae against cancer cell invasion was evaluated through MMP inhibitory effect in HT1080 fibrosarcoma cells in vitro. I. okamurae crude extract was fractionated with organic solvents, namely water (H₂O), n-butanol (n-BuOH), 85% aqueous methanol (85% aq. MeOH), and n-hexane (n-Hex). The non-toxicity of fractions was confirmed by MTT assay. All fractions inhibited the enzymatic activities of MMP-2 and MMP-9 according to gelatin zymography assay. Cell migration was also inhibited by 85% aq. MeOH fraction, significantly. In addition, both gene and protein expressions of MMP-2 and MMP-9, and tissue inhibitor of MMPs (TIMPs) were evaluated by RT-PCR and Western blotting, respectively. Fractions suppressed the mRNA and protein levels of MMP-2 and MMP-9 while elevating the TIMP-1 and TIMP-2, H₂O fraction being the least effective while 85% aq. MeOH fraction the most. Collectively, 85% aq. MeOH fraction from brown algae could be a potential inhibitor of MMPs, suggestively considering presence of poly-unsaturated fatty acids in high amounts.     

Interdomain Flexibility in Full-length Matrix Metalloproteinase-1 (MMP-1)

The presence of extensive reciprocal conformational freedom between the catalytic and the hemopexin-like domains of full-length matrix metalloproteinase-1 (MMP-1) is demonstrated by NMR and small angle x-ray scattering experiments. This finding is discussed in relation to the essentiality of the hemopexin-like domain for the collagenolytic activity of MMP-1. The conformational freedom experienced by the present system, having the shortest linker between the two domains, when compared with similar findings on MMP-12 and MMP-9 having longer and the longest linker within the family, respectively, suggests this type of conformational freedom to be a general property of all MMPs.Matrix metalloproteinases (MMP)² are extracellular hydrolytic enzymes involved in a variety of processes including connective tissue cleavage and remodeling (1–3). All 23 members of the family are able to cleave simple peptides derived from connective tissue components such as collagen, gelatin, elastin, etc. A subset of MMPs is able to hydrolyze more resistant polymeric substrates, such as cross-linked elastin, and partially degraded collagen forms, such as gelatin and type IV collagens (4). Intact triple helical type I–III collagen is only attacked by collagenases MMP-1, MMP-8, and MMP-13 and by MMP-2 and MMP-14 (5–12). Although the detailed mechanism of cleavage of single chain peptides by MMP has been largely elucidated (13–19), little is known about the process of hydrolysis of triple helical collagen. In fact, triple helical collagen cannot be accommodated in the substrate-binding groove of the catalytic site of MMPs (9).All MMPs (but MMP-7) in their active form are constituted by a catalytic domain (CAT) and a hemopexin-like domain (HPX) (20–22). The CAT domain contains two zinc ions and one to three calcium ions. One zinc ion is at the catalytic site and is responsible for the activity, whereas the other metal ions have structural roles. The isolated CAT domains retain full catalytic activity toward simple peptides and single chain polymeric substrates such as elastin, whereas hydrolysis of triple helical collagen also requires the presence of the HPX domain (9, 23–25). It has been shown that the isolated CAT domain regains a small fraction of the activity of the full-length (FL) protein when high amounts of either inactivated full-length proteins or isolated HPX domains are added to the assay solution (9). Finally, it has been shown that the presence of the HPX domain alone alters the CD spectrum of triple helical collagen in a way that suggests its partial unwinding (26, 27). It is tempting to speculate that full-length collagenases attack collagen by first locally unwinding the triple helical structure with the help of the HPX domain and then cleaving the resulting, exposed, single filaments (9, 28).Until 2007, three-dimensional structures of full-length MMPs had been reported only for collagenase MMP-1 (29–31) and gelatinase MMP-2 (32). The structures of the two proteins are very similar and show a compact arrangement of the two domains, which are connected by a short linker (14 and 20 amino acids, respectively). It is difficult to envisage that rigid and compact molecules of this type can interact with triple helical collagen in a way that can lead to first unwinding and then cleavage of individual filaments. It has been recently suggested that such concerted action could occur much more easily if the two domains could enjoy at least a partial conformational independence (9). Slight differences in the reciprocal orientation of the CAT and HPX domains of MMP-1 in the presence (29) and absence (30, 31) of the prodomain were indeed taken as a hint that the two domains could experience relative mobility (29).Two recent solution studies have shown that conformational independence is indeed occurring in gelatinase MMP-9 (33) and elastase MMP-12 (34), whereas the x-ray structure of the latter (34) is only slightly less compact than those of MMP-1 (29–31) and MMP-2 (32). Among MMPs, MMP-9 features an exceptionally long linker (68 amino acid) (33, 35), which in fact constitutes a small domain by itself (the O-glycosylated domain) (33), and therefore, this inspiring observation can hardly be taken as evidence that conformational freedom is a general characteristic of the two-domain MMPs. MMP-12 features a much more normal 16-amino acid linker, thereby making more probable a general functional role for this conformational freedom (34). However, both MMP-9 and MMP-12 retain their full catalytic activity against their substrates even when deprived of the HPX domain (9). Therefore, the question remains of whether conformational freedom is also a required characteristic for those MMPs that are only active as full-length proteins, i.e. collagenases. Interestingly, the three collagenases (MMP-1, MMP-8, and MMP-13) have the shortest linker (14 amino acids) among all MMPs. Demonstrating or negating the presence of conformational freedom in one of these collagenases would therefore constitute a significant step forward to formulate mechanistic hypotheses on their collagenolytic activity.Our recent studies on MMP-12 in solution (34) have shown that a combination of NMR relaxation studies and small angle x-ray scattering (SAXS) is enough to show the presence and the extent of the relative conformational freedom of the two domains of MMPs. Here we apply the same strategy to full-length MMP-1 and show that sizable conformational freedom is indeed experienced even by this prototypical collagenase, although somewhat less pronounced than that observed for MMP-12.     

Matrix Metalloproteinase-13 (MMP-13) Directly and Indirectly Promotes Tumor Angiogenesis

Matrix metalloproteinases (MMPs) are extracellular zinc-dependent endopeptidases involved in the degradation and remodeling of extracellular matrix in physiological and pathological processes. MMPs also have a role in cell proliferation, migration, differentiation, angiogenesis, and apoptosis. We previously identified cancer invasion-related factors by comparing the gene expression profiles between parent and the highly invasive clone of cancer cells. Matrix metalloproteinase-13 (MMP-13) was identified as a common up-regulated gene by cancer invasion-related factors. Although MMP-13 slightly promoted tumor invasion, we found that MMP-13 was involved in tumor angiogenesis. Conditioned medium from MMP-13-overexpressing cells promoted capillary formation of immortalized human umbilical vein endothelial cells. Furthermore, treatment with recombinant MMP-13 protein enhanced capillary tube formation both in vitro and in vivo. MMP-13-promoted capillary tube formation was mediated by activation of focal adhesion kinase and ERK. Interestingly, MMP-13 promoted the secretion of VEGF-A from fibroblasts and endothelial cells. By immunohistochemical analysis, we found a possible correlation between MMP-13 expression and the number of blood vessels in human cancer cases. In summary, these findings suggest that MMP-13 may directly and indirectly promote tumor angiogenesis.     

Soluble glucocorticoid-induced tumor necrosis factor receptor (sGITR) increased MMP-9 activity in murine macrophage

Glucocorticoid induced tumor necrosis factor receptor (GITR), a new TNFR family, increased production of matrix matalloproteinase (MMP-9) in murine macrophages. Murine macrophages produced a band of gelatinolytic activity at 100 kDa when stimulated for 18 h with soluble GITR. MMP-9 was identified by gelatin zymography and Western blot. Previous results demonstrated that murine macrophages express GITR and GITR ligand constitutively. Induction of MMP-9 was synergistic with co-treatment of INF-gamma. MMPs could play a critical role in progression and promotion of tissue injury after inflammation stimulated by GITR/ligand system.     

Imbalances between Matrix Metalloproteinases (MMPs) and Tissue Inhibitor of Metalloproteinases (TIMPs) in Maternal Serum during Preterm Labor

Background

Matrix metalloproteinases (MMPs) are involved in remodeling of the extracellular matrix (ECM) during pregnancy and parturition. Aberrant ECM degradation by MMPs or an imbalance between MMPs and their tissue inhibitors (TIMPs) have been implicated in the pathogenesis of preterm labor, however few studies have investigated MMPs or TIMPs in maternal serum. Therefore, the purpose of this study was to determine serum concentrations of MMP-3, MMP-9 and all four TIMPs as well as MMP:TIMP ratios during term and preterm labor.

Methods

A case control study with 166 singleton pregnancies, divided into four groups: (1) women with preterm birth, delivering before 34 weeks (PTB); (2) gestational age (GA) matched controls, not in preterm labor; (3) women at term in labor and (4) at term not in labor. MMP and TIMP concentrations were measured using Luminex technology.

Results

MMP-9 and TIMP-4 concentrations were higher in women with PTB vs. GA matched controls (resp. p = 0.01 and p<0.001). An increase in MMP-9:TIMP-1 and MMP-9:TIMP-2 ratio was observed in women with PTB compared to GA matched controls (resp. p = 0.02 and p<0.001) as well as compared to women at term in labor (resp. p = 0.006 and p<0.001). Multiple regression results with groups recoded as three key covariates showed significantly higher MMP-9 concentrations, higher MMP-9:TIMP-1 and MMP-9:TIMP-2 ratios and lower TIMP-1 and -2 concentrations for preterm labor. Significantly higher MMP-9 and TIMP-4 concentrations and MMP-9:TIMP-2 ratios were observed for labor.

Conclusions

Serum MMP-9:TIMP-1 and MMP-9:TIMP-2 balances are tilting in favor of gelatinolysis during preterm labor. TIMP-1 and -2 concentrations were lower in preterm gestation, irrespective of labor, while TIMP-4 concentrations were raised in labor. These observations suggest that aberrant serum expression of MMP:TIMP ratios and TIMPs reflect pregnancy and labor status, providing a far less invasive method to determine enzymes essential in ECM remodeling during pregnancy and parturition.     

MMP-9 and MMP-2 gelatinases and TIMP-1 and TIMP-2 inhibitors in breast cancer: correlations with prognostic factors

The goal of our study was to analyse the prognostic values for some matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in breast cancer. We evaluated the activity and the expression levels of MMP-9, MMP-2, TIMP-1 and TIMP-2 in malignant versus benign fresh breast tumor extracts. For this purpose, gelatinzymography, immunoblotting and ELISA were used to analyse the activity and expression of MMPs and TIMPs. We found that MMP-9 expression level and activity are increased in malignant tumors. In addition, MMP-9/TIMP-1 and MMP-2/TIMP-2 ratio values obtained by us were significantly different in malignant tumors compared to benign tumors. We suggest that the abnormal MMP-9/TIMP-1 balance plays a role in the configuration of breast invasive carcinoma of no special type and also in tumor growth, while altered MMP-2/TIMP-2 ratio value could be associated with lymph node invasion and used as a prognostic marker in correlation with Nottingham Prognostic Index. Finally, we showed that in malignant tumors high expression of estrogen receptors is associated with enhanced activity of MMP-2 and increased bcl- 2 levels, while high expression of progesterone receptors is correlated with low TIMP-1 protein levels.     

Effect of Thrombin on Human Amnion Mesenchymal Cells,Mouse Fetal Membranes,and Preterm Birth

Here, we investigated the effects of thrombin on matrix metalloproteinases (MMPs) and prostaglandin (PG) synthesis in fetal membranes. Thrombin activity was increased in human amnion from preterm deliveries. Treatment of mesenchymal, but not epithelial, cells with thrombin resulted in increased MMP-1 and MMP-9 mRNA and enzymatic activity. Thrombin also increased COX2 mRNA and PGE₂ in these cells. Protease-activated receptor-1 (PAR-1) was localized to amnion mesenchymal and decidual cells. PAR-1-specific inhibitors and activating peptides indicated that thrombin-induced up-regulation of MMP-9 was mediated via PAR-1. In contrast, thrombin-induced up-regulation of MMP-1 and COX-2 was mediated through Toll-like receptor-4, possibly through thrombin-induced release of soluble fetal fibronectin. In vivo, thrombin-injected pregnant mice delivered preterm. Mmp8, Mmp9, and Mmp13, and PGE₂ content was increased significantly in fetal membranes from thrombin-injected animals. These results indicate that thrombin acts through multiple mechanisms to activate MMPs and PGE2 synthesis in amnion.     

Adenylyl cyclase-associated protein-1/CAP1 as a biological target substrate of gelatinase B/MMP-9

Matrix metalloproteinases (MMPs) are classically associated with the turnover of secreted structural and functional proteins. Although MMPs have been shown to process also a kaleidoscope of membrane-associated substrates, little is known about the processing of intracellular proteins by MMPs. Physiological and pathological cell apoptosis, necrosis and tumor lysis by chemotherapy, radiotherapy or immunological cytotoxicity, are examples of conditions in which an overload of intracellular proteins becomes accessible to the action of MMPs. We used a model system of dying human myelomonocytic cells to study the processing of intracellular protein substrates by gelatinase B/MMP-9 in vitro. Adenylyl cyclase-associated protein-1 or CAP1 was identified as a novel and most efficient substrate of gelatinase B/MMP-9. The presence of CAP1 in the extracellular milieu in vivo was documented by analysis of urine of patients with systemic autoimmune diseases. Whereas no active MMP-9 could be detected in urines of healthy controls, all urine samples of patients with clinical parameters of renal failure contained activated MMP-9 and/or MMP-2. In addition, in some of these patients indications of CAP1 cleavage are observed, implying CAP1 degradation in vivo. The high turnover rate of CAP1 by MMP-9, comparable to that of gelatin as the natural extracellular substrate of this enzyme, may be critical to prevent pathological conditions associated with considerable cytolysis.     

Purification and molecular docking study of angiotensin I-converting enzyme (ACE) inhibitory peptides from hydrolysates of marine sponge Stylotella aurantium

Angioteinsin I-converting enzyme (ACE) inhibitory peptide was isolated from marine sponge (Stylotella aurantium) hydrolysate prepared by various hydrolysis enzymes. The peptic hydrolysate exhibited highest ACE inhibitory activity among them and was fractionated into three ranges of molecular weight. The below 5 kDa fraction showed the highest ACE inhibitory activity and was used for subsequent purification steps. The amino acid sequences of the purified peptides were identified to be Tyr-Arg (337.2 Da), and Ile-Arg (287.2 Da). The purified peptides from marine sponge had an IC₅₀ value of 237.2 μM and 306.4 μM, respectively. The molecular docking study revealed that ACE inhibitory activity of the purified peptides was mainly attributed to the hydrogen bond interactions and Pi interaction between the dipeptides and ACE. The results suggest that marine sponge, S. aurantium would be an attractive raw material for the manufacture of anti-hypertensive nutraceutical ingredients.