Evaluation of the radiolabeled boronic acid-based FAP inhibitor MIP-1232 for atherosclerotic plaque imaging.

Research towards the non-invasive imaging of atherosclerotic plaques is of high clinical priority as early recognition of vulnerable plaques may reduce the incidence of cardiovascular events. The fibroblast activation protein alpha (FAP) was recently proposed as inflammation-induced protease involved in the process of plaque vulnerability. In this study, FAP mRNA and protein levels were investigated by quantitative polymerase chain reaction and immunohistochemistry, respectively, in human endarterectomized carotid plaques. A published boronic-acid based FAP inhibitor, MIP-1232, was synthetized and radiolabeled with iodine-125. The potential of this radiotracer to image plaques was evaluated by in vitro autoradiography with human carotid plaques. Specificity was assessed with a xenograft with high and one with low FAP level, grown in mice. Target expression analyses revealed a moderately higher protein level in atherosclerotic plaques than normal arteries correlating with plaque vulnerability. No difference in expression was determined on mRNA level. The radiotracer was successfully produced and accumulated strongly in the FAP-positive SK-Mel-187 melanoma xenograft in vitro while accumulation was negligible in an NCI-H69 xenograft with low FAP levels. Binding of the tracer to endarterectomized tissue was similar in plaques and normal arteries, hampering its use for atherosclerosis imaging.

Fingerprinting of gelatinase subtypes for different topographic regions on non-retaining placenta of Holstein cows.

The contribution of matrix metalloproteinases (MMP) to timely discharge of the placenta from bovine uterus at parturition is yet inconclusive, partly because of the presence of multiple MMP forms in situ. In the current study, the expression of different gelatinase subtypes on non-retaining placentas of Holstein cows was fingerprinted by using gelatin zymography. Different topographic regions on the placenta were measured separately, including the placentome-like structure and the fetal and maternal sides of interplacentomal placenta, all sampled from the central and peripheral areas of the placenta, respectively. The spontaneously ruptured umbilical cords were cross-sectioned as fetus end, middle and placenta end also for separate measurement. Body fluids including blood samples from the parturient cows, their neonatal calves and umbilical cord, as well as fetal fluids and the first colostrum were measured concomitantly. Results showed multiple forms of gelatinases subtypes in the placenta tissues and body fluids, including neutrophil gelatinase-associated lipocalin (NGAL)-MMP-9 complex, both the latent and active forms of MMP-2 and MMP-9; of them, the latent forms were much more abundantly and frequently expressed than the active forms. NGAL-MMP-9 complex was more prevalently present in the body fluids than in the placenta tissues. No distinguishable pattern of the expression of any gelatinase subtype was observed among the placentome-like structure, interplacentomal placenta and umbilical cord, or between fetal and maternal sides. Nonetheless, for interplacentomal placenta, proMMP-9 expression was higher in the central than in the peripheral area. In addition, proMMP-2 expression was higher in the rupture end (fetus end) than the placenta end of the umbilical cord. In conclusion, the current validated gelatin zymography detected a gradient proMMP-9 expression on the non-retaining placenta of cows in reverse to the proximity to the umbilical insertion point, and a gradient proMMP-2 expression on a section of the umbilical cord in reverse to the proximity to the rupture site, suggesting roles played by gelatinases in normal discharge of the placenta at term.

Effect of a nutrient mixture on matrix metalloproteinase-9 dimers in various human cancer cell lines.

Strong clinical and experimental evidence demonstrates association of elevated levels of matrix metalloproteinase MMP-9 with cancer progression, metastasis and shortened patient survival, as it plays a key role in tumor cell invasion and metastasis by digesting the basement membrane and ECM components. MMP-9 is secreted in both the monomeric and dimeric form. Although there is little research on MMP-9 dimers, some studies have shown the dimer to be associated with more aggressive tumor progression. Our objective was to study the relative secretion patterns of MMP-9 monomer and dimer in a variety of cancer cell lines and the effect of a nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract on MMP-9 secretion. The cancer cell lines were grown in their respective media, supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 µg/ml) in 24-well tissue culture plates. At near confluence, the cells were treated with NM at 0,10, 50, 100, 500 and 1000 µg/ml. Parallel sets of cultures were treated with PMA (100 ng/ml) for induction of MMP-9. Cell MMP-9 secretion was assayed by gelatinase zymography. MMP-9 dimer secretion patterns of cancer cells fell into different categories. We observed no MMP-9 dimer in prostate DU-145 and PC-3, pancreatic MIA-Pa-Ca2, colon HCT-116, bladder T-24, head and neck FaDu, glioblastoma A-172, T-98 and LN-18 and leukemia HL-60, Jurkat, and Raji cell lines. MMP-dimer secretion only with PMA induction was seen in breast MCF-7 and MDA-MB-231, uterine SK-UT-1, lung A-549, tongue SC-25, melanoma A2058, osteosarcoma U-2OS, rhabdomyosarcoma, fibrosarcoma HT-1080, chondrosarcoma SW-1350 and liposarcoma SW-872. Cervical HeLa and DoTc 2 4510, renal 786-0 and HCC SK-Hep-1 cells exhibited MMP-9 dimer without PMA treatment and increased secretion with PMA treatment. Sarcomas had the highest levels of MMP-9 monomer and dimer with and without PMA among these cancer cell lines. Cervical, uterine and male breast cancer cell lines showed the next highest levels of MMP-9, followed by breast cancer cell lines. Melanoma, renal, lung, head and neck and HCC showed lower levels and prostate, glioblastoma, bladder and leukemia cell lines the lowest. NM showed dose-dependent inhibition of MMP-9 monomer and dimer in all cell lines tested. In conclusion, high MMP-9 and dimer secretion levels correlated with the most aggressive cancer cell lines. NM was effective in inhibiting MMP-9 and dimer secretion in all cell lines tested, suggesting its therapeutic potential as an antimetastatic agent.

Fisetin inhibits matrix metalloproteinases and reduces tumor cell invasiveness and endothelial cell tube formation.

Matrix metalloproteinases (MMPs) play an important role in tissue remodeling during normal physiological situations and pathological implications such as tumor invasion and metastasis. MMP inhibitors were screened from extracts of medicinal herbs by an enzymatic assay using the MMP-14 catalytic domain. Among samples tested, a methanol extract of the root of Dalbergia odorifera T. CHEN (Leguminosae) showed the strongest inhibitory activity. The inhibitory component was purified through fractionation methods and identified as fisetin, abundant in many fruits and vegetables. In addition to inhibition of MMP-14, fisetin inhibits MMP-1, MMP-3, MMP-7, and MMP-9, more efficiently than a naturally occurring MMP inhibitor tetracycline. Fisetin dose-dependently inhibits proliferation of fibrosarcoma HT-1080 cells and human umbilical vascular endothelial cells (HUVECs), MMP-14-mediated activation of proMMP-2 in HT-1080 cells, invasiveness of HT-1080 cells, and in vitro tube formation of HUVECs. Therefore, fisetin could be valuable as a chemopreventive agent against cancer and a lead compound for development of therapeutic MMP inhibitors.

Fibrogenic and redox-related but not proinflammatory genes are upregulated in Lewis rat model of chronic silicosis.

Silicosis, a fibrotic granulomatous lung disease, may occur through accidental high-dose or occupational inhalation of silica, leading to acute/accelerated and chronic silicosis, respectively. While chronic silicosis has a long asymptomatic latency, lung inflammation and apoptosis are hallmarks of acute silicosis. In animal models, histiocytic granulomas develop within days after high-dose intratracheal (IT) silica instillation. However, following chronic inhalation of occupationally relevant doses of silica, discrete granulomas resembling human silicosis arise months after the final exposure without significant lung inflammation/apoptosis. To identify molecular events associated with chronic silicosis, lung RNA samples from controls or subchronic silica-exposed rats were analyzed by Affymetrix at 28 wk after silica exposures. Results suggested a significant upregulation of 144 genes and downregulation of 7 genes. The upregulated genes included complement cascade, chemokines/chemokine receptors, G-protein signaling components, metalloproteases, and genes associated with oxidative stress. To examine the kinetics of gene expression relevant to silicosis, quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), Luminex-bead assays, Western blotting, and/or zymography were performed on lung tissues from 4 d, 28 wk, and intermediate times after subchronic silica exposure and compared with 14-d acute silicosis samples. Results indicated that genes regulating fibrosis (secreted phosphoprotein-1, Ccl2, and Ccl7), redox enzymes (superoxide dismutase-2 and arginase-1), and the enzymatic activities of matrix metalloproteinases 2 and 9 were upregulated in acute and chronic silicosis models. However, proinflammatory cytokines were strongly upregulated only in acute silicosis. Thus, inflammatory cytokines are associated with acute but not chronic silicosis. Data suggest that genes regulating fibrosis, oxidative stress, and metalloproteases may contribute to both acute and chronic silicosis.

Crocetin inhibits invasiveness of MDA-MB-231 breast cancer cells via downregulation of matrix metalloproteinases.

Crocetin is a carotenoid dicarboxylic acid which, in nature, is esterified with glucose or gentiobiose units forming the crocins, abundant components of saffron (a spice with many reputed medicinal uses). We have previously reported that saffron, crocins and crocetin inhibit breast cancer cell proliferation. In order to further study the effect of crocetin on breast cancer cells, we used the highly invasive MDA-MB-231 cells and measured the viability with the WST-1 assay and the invasiveness through a reconstituted basement membrane. After 24 h incubation, crocetin significantly inhibited not only proliferation but also invasion at 1 and 10 µM. Cancer invasiveness and metastasis are associated with the expression of matrix metalloproteinases (MMPs). In order to study the molecular changes of MMP expression that might accompany the observed crocetin effects, gene expression of MMPs was studied by RT-PCR, whereas protein expression and gelatinolytic activity were determined with Western blotting and zymography, respectively. The gene and protein expression of pro-MT1-MMP and pro-MT2-MMP were greatly attenuated by both crocetin and all- TRANS-retinoic acid (ATRA, used as control). Incubation with 10 µM crocetin for 24 h in serum-free conditions reduced pro-MMP-9 activity and pro-MMP-2/MMP-2 protein levels. When cultured in media with sera 2 and 5 %, crocetin at 10 µΜ also reduced gelatinase activity. The above findings show that crocetin, the main metabolite of crocins, inhibits MDA-MB-231 cell invasiveness via downregulation of MMP expression.

Calmodulin inhibitors trigger the proteolytic processing of membrane type-1 matrix metalloproteinase, but not its shedding in glioblastoma cells.

Most transmembrane proteins are subjected to limited proteolysis by cellular proteases, and stimulation of cleavage of membrane proteins by calmodulin (CaM) inhibitors was recently shown. The present study investigated the ability of several CaM inhibitors to induce the proteolytic cleavage of the membrane type-1 matrix metalloproteinase (MT1-MMP) from the cell surface of highly invasive U-87 glioblastoma cells. Although no shedding of a soluble MT1-MMP form was induced by CaM inhibitors in the conditioned media, we showed that these inhibitors induced MT1-MMP proteolytic processing to the 43 kDa membrane-bound inactive form that was not correlated with an increase in proMMP-2 activation but rather with an increase in tissue inhibitor of MMPs (TIMP)-2 expression levels. Moreover, this proteolytic processing was sensitive to marimastat suggesting the involvement of MMPs. Interestingly, CaM inhibitors antagonized concanavalin A- and cytochalasin D-induced proMMP-2 activation, and affected the cytoskeletal actin organization resulting in the loss of migratory potential of U-87 glioblastoma cells. Cytoplasmic tail-truncated MT1-MMP constructs expressed in COS-7 cells were also affected by CaM inhibitors suggesting that these inhibitors stimulated MT1-MMP proteolytic processing by mechanisms independent of the CaM-substrate interaction. We also propose that TIMP-2 acts as a negative regulator of MT1-MMP-dependent activities promoted by the action of CaM inhibitors in U-87 glioblastoma cells.

Gelatinases A and B and tissue inhibitors of metalloproteinases 1, 2, and 3 during in vivo and in vitro decidualization of rat endometrial stromal cells.

An important event during decidualization is the remodeling of the extracellular matrix, an event controlled by the balance of matrix metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). A putative regulator of decidualization is prostaglandin E2 (PGE2). The present study shows that endometrial mRNA levels for TIMPs 1, 2, and 3 were increased while gelatinase A levels remained unchanged and gelatinase B levels decreased during oil-induced decidualization. The production of TIMPs 1, 2, and 3 and gelatinases A and B during in vitro decidualization was examined, as was the role of PGE2 as a regulator. Ovariectomized rats were given a regimen of estrogen and progesterone, which sensitized their uteri for decidualization, at which time endometrial stromal cells were isolated and cultured in serum-free conditions for 72 h. Northern blot analyses indicated the presence of the mRNAs for TIMPs and gelatinases, while reverse zymography and zymography showed the presence of their proteins. PGE2 decreased mRNA levels for TIMP-1 and gelatinase A but had no effect on gelatinase B or TIMPs 2 and 3. Indomethacin had no effect on any of the transcripts. These data indicate that rat endometrial stromal cells undergoing decidualization in vitro secrete gelatinases and TIMPs, and suggest that PGE2 may play a role in regulating tissue remodeling during decidualization.

Transcriptional suppression of matrix metalloproteinase-2 gene expression in human astroglioma cells by TNF-alpha and IFN-gamma.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that function in the turnover of extracellular matrix components during development. In addition, MMPs also contribute to pathological conditions associated with inflammation, angiogenesis, and tumor invasion. A 72-kDa type IV collagenase, also referred to as gelatinase A or MMP-2, has been proposed to potentiate the invasion and metastasis of malignant tumors. In particular, MMP-2 activity has been shown to constitute an important component of human astroglioma invasion. We investigated the influence of various cytokines, both proinflammatory and immunosuppressive, on MMP-2 gene expression in two human astroglioma cell lines (U251-MG and CRT). Our results indicate that the cell lines constitutively express high levels of MMP-2 mRNA, protein, and bioactivity as assessed by ribonuclease protection assay, immunoblotting, and zymography assays, respectively. The proinflammatory cytokines TNF-alpha and IFN-gamma individually can inhibit constitutive MMP-2 expression, and function in an additive manner for near-complete inhibition of MMP-2 expression. Inhibition of MMP-2 mRNA levels by TNF-alpha and IFN-gamma is not due to destabilization of the MMP-2 message; rather, inhibition is mediated at the transcriptional level. Furthermore, TNF-alpha/IFN-gamma inhibition of MMP-2 expression results in decreased invasiveness of the human astroglioma cells through an extracellular matrix. These results raise the possibility that TNF-alpha and IFN-gamma may have beneficial effects in attenuating astroglioma invasive properties.

Cloning of murine membrane-type-1-matrix metalloproteinase (MT-1-MMP) and its metanephric developmental regulation with respect to MMP-2 and its inhibitor.

BACKGROUND: Extracellular matrix macromolecules regulate morphogenesis of embryonic organs, and are developmentally regulated. Their expression and turnover is regulated by matrix metalloproteinases (MMPs). Recently, an epithelial cell "membrane" associated metalloproteinase (MT-1-MMP) has been identified that acts as an activator of a "secreted" MMP-2, and is produced by mesenchymal fibroblasts. The activity of MMP-2 is inhibited by a "soluble" tissue inhibitor of MMP-2, TIMP-2. The role of MT-1-MMP in renal development is unknown. METHODS: MT-1-MMP was cloned from embryonic mouse kidney cDNA library, and its spatio-temporal distribution during development in the context of MMP-2 and tissue inhibitor of metalloproteinase-2 (TIMP-2) was studied. RESULTS: The cloned MT-1-MMP exhibited approximately 86% nucleotide sequence homology with human MT-1-MMP, and had a catalytic domain and a zinc binding site preceded by a RRKR furin recognition motif. A approximately 4.5 Kb MT-1-MMP mRNA transcript was detected, and its expression was developmentally regulated. A parallel developmental regulation of MMP-2 mRNA expression was also observed. TIMP-2 expression was also developmentally regulated, but lagged behind MT-1-MMP and MMP-2. By in situ hybridization, MT-1-MMP mRNA was seen to be confined to ureteric bud epithelia, and was absent in the mesenchyme, while MMP-2 was confined to the mesenchyme. MT-1-MMP protein expression was seen on ureteric bud epithelia, induced mesenchyme and nascent nephrons, and it was highest during mid gestation. Similar spatio-temporal expressions of MMP-2 and TIMP-2 proteins were observed. CONCLUSIONS: mRNAs of MT-MMP-1 and MMP-2 are expressed in the respective epithelial and mesenchymal compartments, while their proteins are co-expressed in the epithelia suggest that MT-1-MMP and MMP-2, in conjunction with TIMP-2, may be involved in paracrine/juxtacrine epithelial:mesenchymal interactions during metanephrogenesis.

UV irradiation induces the expression of gelatinases in human skin in vivo.

UV irradiation results in marked changes in skin connective tissue, such as degeneration of collagen, and abnormal elastosis. The mechanism of connective tissue damage by UV has not been clarified in detail. In the present study the mechanism of actinic damage was studied by assaying gelatinases, 72-kDa (MMP-2) and 92-kDa (MMP-9), from suction blister fluids induced on patients who had received either UVB or PUVA treatments. The results indicate that both UVB and PUVA increase the levels of gelatinases in human skin. By in situ hybridization, it was also possible to show that UV irradiation induced increased levels of gelatinase mRNAs in fibroblasts. Furthermore, in samples from severe actinic damage, gelatinase mRNAs were abundantly present, suggesting that gelatinases may contribute to photodamage.

Expression of 92 kD type IV collagenase/gelatinase B in human osteoclasts.

The digestion of type I collagen is an essential step in bone resorption. It is well established that osteoclasts solubilize the mineral phase of bone during the resorptive process, but the mechanism by which they degrade type I collagen, the major proteinaceous component of bone, is controversial. Differential screening of a human osteoclastoma cDNA library was performed to characterize genes specifically expressed in osteoclasts. A large number of cDNA clones obtained by this procedure were found to represent 92 kD type IV collagenase (gelatinase B; MMP-9, EC 3.4.24.35), as well as tartrate-resistant acid phosphatase. In situ hybridization localized mRNA for gelatinase B to multinucleated giant cells in human osteoclastomas. Gelatinase B immunoreactivity was demonstrated in giant cells from eight of eight osteoclastomas, osteoclasts in normal bone, and osteoclasts of Paget's disease by use of a polyclonal antiserum raised against a synthetic gelatinase B peptide. In contrast, no immunoreactivity for 72 kD type IV collagenase (gelatinase A; MMP-2, EC 3.4.24.24), which is the product of a separate gene, was detected in osteoclastomas or normal osteoclasts. We propose that the 92 kD type IV collagenase/gelatinase B plays an important role in the resorption of collagen during bone remodeling.