Human Tissue Inhibitor Of Metalloproteinases Research Articles

Up-regulation of tissue inhibitor of metalloproteinase-2 promotes SHI-1 cell invasion in nude mice

The role of tissue inhibitor of metalloproteinase-2 (TIMP-2) in extramedullary infiltration of acute leukemia is unclear. We demonstrated in our previous study that the up-regulation of TIMP-2 promoted SHI-1 cell invasion in vitro. We investigated in the present study whether TIMP-2 would have the same effect in vivo. A retroviral vector carrying human TIMP-2 cDNA was constructed and transfected into SHI-1 cells. Three subclone cells (S1, S2 and S3) that highly expressed TIMP-2 were selected to establish nude mouse models of acute leukemia. Times of leukemic onset in mice of S1, S2 and S3 groups were all earlier than that of the SHI-1 group, whereas the survival times of S1, S2 and S3 groups were all shorter than that of the SHI-1 group (p < 0.05). Histopathological results demonstrated severe leukemic infiltration in numerous organs in each group. Reverse transcription polymerase chain reaction (RT-PCR) assay showed that several organs expressed the MLL/F6 fusion gene. Moreover, the numbers of organs infiltrated by leukemic cells in S1, S2 and S3 groups were more than those in the SHI-1 group (p < 0.05). Up-regulating TIMP-2 expression enhanced SHI-1 cell invasion in nude mice and resulted in more severe leukemia infiltration. This phenomenon suggests that targeted therapy with TIMP-2 for acute leukemia should be performed with prudence.

PEGylation Extends Circulation Half-Life While Preserving In Vitro and In Vivo Activity of Tissue Inhibitor of Metalloproteinases-1 (TIMP-1)

Excess proteolytic activity of matrix metalloproteinases (MMPs) contributes to the development of arthritis, cardiovascular diseases and cancer progression, implicating these enzymes as therapeutic targets. While many small molecule inhibitors of MMPs have been developed, clinical uses have been limited, in part by toxicity and off-target effects. Development of the endogenous tissue inhibitors of metalloproteinases (TIMPs) as recombinant biopharmaceuticals represents an alternative therapeutic approach; however, the short plasma half-life of recombinant TIMPs has restricted their potential in this arena. To overcome this limitation, we have modified recombinant human TIMP-1 (rhTIMP-1) by PEGylation on lysine residues. We analyzed a mixture of mono- and di-PEGylated rhTIMP-1 species modified by attachment of 20 kDa mPEG chains (PEG20K-TIMP-1), as confirmed by SELDI-TOF mass spectrometry. This preparation retained complete inhibitory activity toward the MMP-3 catalytic domain and partial inhibitory activity toward full length MMP-9. Pharmacokinetic evaluation showed that PEGylation extended the plasma half-life of rhTIMP-1 in mice from 1.1 h to 28 h. In biological assays, PEG20K-TIMP-1 inhibited both MMP-dependent cancer cell invasion and tumor cell associated gelatinase activity. Overall these results suggest that PEGylated TIMP-1 exhibits improved potential for development as an anti-cancer recombinant protein therapeutic, and additionally may offer potential for clinical applications in the treatment of other diseases.

Phage Display of Tissue Inhibitor of Metalloproteinases-2 (TIMP-2): IDENTIFICATION OF SELECTIVE INHIBITORS OF COLLAGENASE-1 (METALLOPROTEINASE 1 (MMP-1))

Tissue inhibitor of metalloproteinases-2 (TIMP-2) is a broad spectrum inhibitor of the matrix metalloproteinases (MMPs), which function in extracellular matrix catabolism. Here, phage display was used to identify variants of human TIMP-2 that are selective inhibitors of human MMP-1, a collagenase whose unregulated action is linked to cancer, arthritis, and fibrosis. Using hard randomization of residues 2, 4, 5, and 6 (L1) and soft randomization of residues 34-40 (L2) and 67-70 (L3), a library was generated containing 2 × 10(10) variants of TIMP-2. Five clones were isolated after five rounds of selection with MMP-1, using MMP-3 as a competitor. The enriched phages selectively bound MMP-1 relative to MMP-3 and contained mutations only in L1. The most selective variant (TM8) was used to generate a second library in which residues Cys(1)-Gln(9) were soft-randomized. Four additional clones, selected from this library, showed a similar affinity for MMP-1 as wild-type TIMP-2 but reduced affinity for MMP-3. Variants of the N-terminal domain of TIMP-2 (N-TIMP-2) with the sequences of the most selective clones were expressed and characterized for inhibitory activity against eight MMPs. All were effective inhibitors of MMP-1 with nanomolar K(i) values, but TM8, containing Ser(2) to Asp and Ser(4) to Ala substitutions, was the most selective having a nanomolar K(i) value for MMP-1 but no detectable inhibitory activity toward MMP-3 and MMP-14 up to 10 μM. This study suggests that phage display and selection with other MMPs may be an effective method for discovering tissue inhibitor of metalloproteinase variants that discriminate between specified MMPs as targets.

Generation of antibodies recognizing an aberrant glycoform of human tissue inhibitor of metalloproteinase-1 (TIMP-1) using decoy immunization and phage display

Aberrant glycosylation of human tissue inhibitor of metalloproteinase-1 (TIMP-1) by N-acetylglucosaminyltransferase-V (GnT-V) was previously reported to be related to cancer progression. Here, we report on the antibodies recognizing the structural features initiated by an addition of N-linked β(1,6)- N-acetylglucosamine (GlcNAc) branch by GnT-V on TIMP-1. Two glycoforms of TIMP-1, TIMP1-L produced in Lec4 cells without GnT-V activity and TIMP1-B in GnT-V overexpressing transfectant cells, were purified from culture supernatant and used for generation of antibodies. TIMP1-L was injected in the left hind footpad of mice as decoy and TIMP1-B in the right hind footpad as immunogen. Phage-displayed scFv library was constructed from the B cells retrieved from the right popliteal lymph nodes and subjected to panning and screening. Phage ELISA of individual clones revealed the scFv clones with preferential binding activity to TIMP1-B, and they were converted into an scFv-Fc format for further characterization of binding specificity. Glycan binding assay of an antibody, 1-9F, revealed its differential specificity toward an extension of glycan structure initiated with β(1,6)-GlcNAc linkage and terminally decorated with a sialic acid. This study demonstrates feasibility of a new strategy combining decoy immunization with phage display for the efficient generation of antibodies tracking down structural features of different glycoforms.

Membrane Type 1 Matrix Metalloproteinase Detection in Tumors, Using the Iodinated Endogenous [123I]-Tissue Inhibitor 2 of Metalloproteinases as Imaging Agent

Matrix metalloproteinases (MMPs) are principal participants in tumor development. In addition to serve as a useful biochemical marker, MMP expression may also provide a target for the diagnostic in vivo imaging of tumors, using a radiolabeled inhibitor. This study investigates the use of membrane type 1 (MT1)-MMP as target for in vivo tumor diagnosis. Specific binding of the endogenous tissue inhibitor of metalloproteinase-2 (TIMP-2) to MT1-MMP has been previously described. In this study, biodistribution and imaging experiments were performed on MT1-MMP-overexpressing (S.1.5) and control (C.IV.3) tumor-inoculated mice using [(123)I]-recombinant human TIMP-2 (rhTIMP-2) as radioligand and [(123)I]-rhTIMP-1 as control. The expression profile was controlled in vitro and on tumor extracts. rhTIMP-2 as well as rhTIMP-1 were labeled using the Iodogen method and characterized. Biodistribution of [(123)I]-rhTIMP-2 showed a tumor uptake of 2.87% ± 1.58% ID/g at 3 hours postinjection in S.1.5. Tumor values of [(123)I]-rhTIMP-1 and [(123)I]-rhTIMP-2 evaluated in S.1.5 and C.IV.3, respectively, were significantly lower. Planar imaging revealed significant uptake of [(123)I]-rhTIMP-2 in S.1.5 compared with contralateral background areas. This could not be observed in C.IV.3 and with [(123)I]-rhTIMP-1 in S.1.5. All tumors were well established (200-800 mg). These results suggest that rhTIMP-2 holds potential for development of radiotracers for in vivo imaging in overexpressing MT1-MMP but not in similar tumors that do not express this protease.

Enhancement of Procollagen Biosynthesis by p180 through Augmented Ribosome Association on the Endoplasmic Reticulum in Response to Stimulated Secretion

A coiled-coil microtubule-bundling protein, p180, was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported a novel role for p180 in the trans-Golgi network (TGN) expansion following stimulated collagen secretion. Here, we show that p180 plays a key role in procollagen biosynthesis and secretion in diploid fibroblasts. Depletion of p180 caused marked reductions of secreted collagens without significant loss of the ER membrane or mRNA. Metabolic labeling experiments revealed that the procollagen biosynthetic activity was markedly affected following p180 depletion. Moreover, loss of p180 perturbs ascorbate-stimulated de novo biosynthesis mainly in the membrane fraction with a preferential secretion defect of large proteins. At the EM level, one of the most prominent morphological features of p180-depleted cells was insufficient ribosome association on the ER membranes. In contrast, the ER of control cells was studded with numerous ribosomes, which were further enhanced by ascorbate. Similarly biochemical analysis confirmed that levels of membrane-bound ribosomes were altered in a p180-dependent manner. Taken together, our data suggest that p180 plays crucial roles in enhancing collagen biosynthesis at the entry site of the secretory compartments by a novel mechanism that mainly involves facilitating ribosome association on the ER.

TIMP-1 Status Measured by Immunohistochemistry in 268 ER Positive Postmenopausal High-Risk Breast Cancer Patients Treated with Adjuvant Tamoxifen: Association to Recurrence Free Survival.

Abstract Background: A proportion of ER positive breast cancer patients do not benefit from adjuvant tamoxifen treatment. Tamoxifen has been shown to induce apoptosis in human breast cancer cells in vitro and Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) protects cancer cells against apoptosis. We therefore raised the hypothesis that ER positive breast cancer cells with high TIMP-1 immunoreactivity would show reduced benefit to tamoxifen treatment.Material and methods: Tumor tissue from 268 high-risk postmenopausal ER positive breast cancer patients was used for this study. All patients underwent surgery for primary invasive breast cancer. The majority of the patients were lymph node positive: 92% (246/268). Patients were allocated to 1, 2 or 5 years of adjuvant tamoxifen as mono-therapy. Recurrent disease was diagnosed in 33% (89/268) of the patients. Median time to recurrence was 3.1 years and the median time of follow-up was 12.4 years. Archival formalin-fixed and paraffin-embedded primary tumor tissue was used to generate tissue microarrays comprising two, 2 mm cores from each tumor. TIMP-1 immunoreactivity was evaluated using the mouse monoclonal antibody (clone VT7) raised against recombinant human TIMP-1. This antibody exclusively reacts with TIMP-1. A central assessment of TIMP-1 status was performed. Tumors were analysed by bright field microscopy and scored semi-quantitatively as 0 - 3 with regard to the number of stained tumour cells and intensity of the staining. An index combining the number of positive tumor cells and staining intensity was created: low (0-2), median (3-4) and high expression (5-6).Results: IHC was successfully performed in 266 cases. TIMP-1 IHC negative cases were observed in 39% (105/266) of the tumors and 61% (161/266) were TIMP-1 IHC positive. The index combining the number of positive tumor cells and staining intensity showed low expression in 59% (157/266), median expression in 24% (65/266) and high expression in 17% (44/266) of the tumors. TIMP-1 negativity and low TIMP-1 index showed no significant association with tumor grade, tumor size or lymph node status. When performing Kaplan-Meier plots, TIMP-1 expression was not significantly associated with recurrence free survival in univariate analyses (p = 0.1).Conclusion: This study did not confirm our hypothesis that TIMP-1 immunoreactivity in ER positive breast cancer cells are associated with reduced benefit from adjuvant tamoxifen treatment. This is partly in contrast with prior studies on endocrine therapy of metastatic breast cancer (Lipton et al 2008) where high serum TIMP-1 was associated with reduced response to endocrine therapy. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2025.

Regulation of soluble vascular endothelial growth factor receptor 1 secretion from human endothelial cells by tissue inhibitor of metalloproteinase 1

Vascular endothelial growth factor (VEGF) and its soluble receptor (sVEGFR-1) are key regulators in human ovarian angiogenesis. Produced by granulosa and ovarian theca interna cells, VEGF promotes blood vessel growth during follicular development and corpus luteum formation, whereas sVEGFR-1, which is secreted by endothelial cells, functions as an antagonist to VEGF activity by binding it. In order to gain further insights into the regulatory mechanisms of ovarian angiogenesis, the aim of the present study was to analyze the influence of tissue inhibitor of metalloproteinase 1 (TIMP-1), which is actively involved in the degradation and remodeling of the extracellular matrix, on sVEGFR-1 secretion of cultured human umbilical vein endothelial cells. sVEGFR-1 production was determined in the culture supernatant by Sandwich-ELISA. We showed that TIMP-1 produced by human granulosa cells and recombinant human TIMP-1 both significantly increased the production of sVEGFR-1 in endothelial cells. Also, the down-regulation of TIMP-1 expression by RNA interference resulted in a significant reduction of endothelial sVEGFR-1 secretion into the culture medium. Furthermore, TIMP-1 weakly inhibited proliferation of VEGF-stimulated endothelial cells. In conclusion, our results provide evidence that TIMP-1 increases the production of sVEGFR-1 in endothelial cells and thus may reduce VEGF bioavailability, leading to reduced blood vessel growth in the ovary.

Smad signaling pathway is a pivotal component of tissue inhibitor of metalloproteinases-3 regulation by transforming growth factor beta in human chondrocytes

Transforming growth factor beta (TGF-β1) promotes cartilage matrix synthesis and induces tissue inhibitor of metalloproteinases-3 (TIMP-3), which inhibits matrix metalloproteinases, aggrecanases and TNF-α-converting enzyme implicated in articular cartilage degradation and joint inflammation. TGF-β1 activates Akt, ERK and Smad2 pathways in chondrocytes. Here we investigated previously unexplored roles of specific Smads in TGF-β1 induction of TIMP-3 gene by pharmacological and genetic knockdown approaches. TGF-β1-induced Smad2 phosphorylation and TIMP-3 protein expression could be inhibited by the Smad2/3 phosphorylation inhibitors, PD169316 and SB203580 and by Smad2-specific siRNA. Specific inhibitor of Smad3 (SIS3) and Smad3 siRNA abolished TGF-β induction of TIMP-3. Smad2/3 siRNAs also down regulated TIMP-3 promoter-driven luciferase activities, suggesting transcriptional regulation. SiRNA-driven co-Smad4 knockdown abrogated TIMP-3 augmentation by TGF-β. TIMP-3 promoter deletion analysis revealed that − 828 deletion retains the original promoter activity while − 333 and − 167 deletions display somewhat reduced activity suggesting that most of the TGF-β-responsive, cis-acting elements are found in the − 333 fragment. Chromatin Immunoprecipitation (ChIP) analysis confirmed binding of Smad2 and Smad4 with the − 940 and − 333 promoter sequences. These results suggest that receptor-activated Smad2 and Smad3 and co-Smad4 critically mediate TGF-β-stimulated TIMP-3 expression in human chondrocytes and TIMP-3 gene is a target of Smad signaling pathway.

Tissue inhibitor of metalloproteinase-1 exacerbated renal interstitial fibrosis through enhancing inflammation

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is associated with renal fibrosis. Furthermore, it is a multi-functional protein, and whether it has other roles in renal fibrosis is unknown. As several inflammatory mediators are substrates of matrix metalloproteinases (MMPs), TIMP-1 might affect renal fibrosis via inflamatory pathways. Plasmids containing the sense and antisense human TIMP-1 sequence were stably transfected into the human kidney proximal tubular epithelial cell line (HKC), MMP-2 and MMP-9 siRNA were transiently transfected into HKC and the transfected cells were stimulated with phorbol 12-myristate 13-acetate (PMA). In vivo, we established unilateral ureteral obstruction (UUO) models by using homozygote human TIMP-1 transgenic mice. The expression of intercellular adhesion molecule-1 (ICAM-1) in transfected cells and F4/80-positive cells in the renal interstitium were examined by indirect immunofluorescence. Protein levels in the cells and UUO models were examined by western blot, and the activities of the gelatinases and TIMP-1 were examined by gelatin zymography and reverse zymography, respectively. After stimulation with PMA, the activities of the gelatinases were decreased, ICAM-1 was upregulated, and soluble ICAM-1 in the supernatant was decreased, in HKC transfected with sense TIMP-1, and ICAM-1 was increased in HKC transfected with MMP-9 siRNA. At 14 days after UUO, it was found that compared with wild-type mice, in transgenic mice, with upregulation of TIMP-1, activities of gelatinases were downregulated, ICAM-1, transforming growth factor-beta1 (TGF-beta1), collagens I and III were upregulated, and the extent of renal fibrosis and infiltration of macrophages was more severe. Overexpression of TIMP-1 could promote renal interstitial fibrosis through the inflammatory pathway, which might be partly induced by upregulating ICAM-1.

Tissue Inhibitor of Metalloproteinase-3 via Oncolytic Herpesvirus Inhibits Tumor Growth and Vascular Progenitors

Malignant solid tumors remain a significant clinical challenge, necessitating innovative therapeutic approaches. Oncolytic viral therapy is a nonmutagenic, biological anticancer therapeutic shown to be effective against human cancer in early studies. Because matrix metalloproteinases (MMP) play important roles in the pathogenesis and progression of cancer, we sought to determine if "arming" an oncolytic herpes simplex virus (oHSV) with an MMP-antagonizing transgene would increase virus-mediated antitumor efficacy. We generated oHSVs that express human tissue inhibitor of metalloproteinases 3 (TIMP3) or firefly luciferase and designated them rQT3 and rQLuc, respectively. We evaluated the antitumor efficacy of these viruses against neuroblastoma and malignant peripheral nerve sheath tumor (MPNST) xenografts. Relative to rQLuc, rQT3-infected primary human MPNST and neuroblastoma cells exhibited equivalent virus replication but increased cytotoxicity and reduced MMP activity. In vivo, rQT3-treated tumors showed delayed tumor growth, increased peak levels of infectious virus, immature collagen extracellular matrix, and reduced tumor vascular density. Remarkably, rQT3 treatment reduced circulating endothelial progenitors, suggesting virus-mediated antivasculogenesis. We conclude that rQT3 enhanced antitumor efficacy through multiple mechanisms, including direct cytotoxicity, elevated virus titer, and reduced tumor neovascularization. These findings support the further development of combined TIMP-3 and oncolytic virotherapy for cancer.

Association of polymorphisms of the MMP-2 and TIMP-2 genes with the risk of endometriosis in North Chinese women

We investigated whether three polymorphisms in the matrix metalloproteinase-2 (MMP-2; -1306C-->T and -735C-->T) and tissue inhibitor of metalloproteinase-2 (TIMP-2; -418G-->C) genes were related to the risk of endometriosis in reproductive-aged women with and without endometriosis. Our results indicate that the TIMP-2 -418C/C homozygote may be a protective factor against the development of endometriosis in North Chinese women.

Tissue Inhibitor of Metalloproteinases-3 Promoter Methylation is an Independent Prognostic Factor for Bladder Cancer

TIMP-3 (tissue inhibitor of metalloproteinases-3) is 1 of 4 members of a family of proteins that were originally classified according to their ability to inhibit matrix metalloproteinases. We analyzed TIMP-3 methylation in 175 urine sediment DNA samples from patients with bladder cancer with well characterized clinicopathological parameters, including patient outcome. We examined urine sediment DNA for aberrant methylation of 9 genes, including TIMP-3, by quantitative fluorogenic real-time polymerase chain reaction. Using an optimal cutoff value by TaqMan(R) quantitation we found that the risk of death was statistically significantly higher in patients with higher TIMP-3 and ARF methylation (HR 1.99, 95% CI 1.12 to 3.27, p = 0.01 and HR 1.66, 95% CI 1.00 to 2.76, p = 0.05, respectively) than in patients without/lower TIMP3 and ARF methylation in urine. A significant correlation was also seen between the risk of death and stage 3 tumor (HR 2.73, 95% CI 1.58 to 4.72, p = 0.003) and metastasis (HR 3.32, 95% CI 1.98 to 5.57, p = 0.0001). Multivariate analysis subsequently revealed that TIMP-3 methylation was an independent prognostic factor for bladder cancer survival with stage and metastasis (p = 0.001 and 0.02, respectively). These results suggest that TIMP-3 promoter methylation could be a clinically applicable marker for bladder cancer progression.

Cloning and expressing a recombinant human tissue inhibitor of metalloproteinase-2 (TIMP-2) in methylotrophic yeast Pichia pastoris and its characterizations

To obtain human tissue inhibitor of metalloproteinase-2 (TIMP-2) cDNA and the secretory expression of TIMP-2 gene in Pichia pastoris, we designed and synthesized a 618 base pairs artificial gene coding for the TIMP-2 with a computer-aided design method using a standard chemical synthesis technique, which was composed of frequently used codons in the highly expressed Pichia pastoris genes. Then the synthetic gene encoding TIMP-2 was checked by means of dideoxynucleotide sequencing. The verified gene of TIMP-2 was cloned to the Escherichia coli-yeast shuttle vector of pPIC9 to construct a recombinant plasmid pPIC9-T2. The plasmid was transformed into GS115 cells of the methylotrophic yeast, Pichia pastoris by electroporation, and we got the expression cell through phenotype selection and induction with methanol. Separation, purification, and bioactivity analysis of the expressed products were performed.

Investigation of Tissue Inhibitor of Metalloproteinases 1 in Plasma from Colorectal Cancer Patients and Blood Donors by Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Early detection of colorectal cancer (CRC) improves patient survival. Plasma tissue inhibitor of metalloproteinases 1 (TIMP-1) measurements by enzyme-linked immunosorbent assay (ELISA) have been suggested as a new method for the early detection of CRC. To further investigate the nature of TIMP-1 in plasma, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI TOF MS) was used. TIMP-1 measurements of plasma from 16 healthy donors and 14 CRC patients were performed using TIMP-1 monoclonal antibody in SELDI TOF MS and ELISA. SELDI TOF MS applying an antibody to TIMP-1 revealed that human plasma TIMP-1 has a mass of 25.1 kDa and exhibits several isoforms. Both methods showed increased plasma TIMP-1 values for cancer patients as compared to healthy individuals. The p values for the separation of the groups were 0.0019 for ELISA and &lt;0.0001 for SELDI TOF MS. CRC did not fundamentally affect the appearance of TIMP-1 as evaluated by SELDI TOF MS.

Investigation of Tissue Inhibitor of Metalloproteinases 1 in Plasma from Colorectal Cancer Patients and Blood Donors by Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Early detection of colorectal cancer (CRC) improves patient survival. Plasma tissue inhibitor of metalloproteinases 1 (TIMP-1) measurements by enzyme-linked immunosorbent assay (ELISA) have been suggested as a new method for the early detection of CRC. To further investigate the nature of TIMP-1 in plasma, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI TOF MS) was used. TIMP-1 measurements of plasma from 16 healthy donors and 14 CRC patients were performed using TIMP-1 monoclonal antibody in SELDI TOF MS and ELISA. SELDI TOF MS applying an antibody to TIMP-1 revealed that human plasma TIMP-1 has a mass of 25.1 kDa and exhibits several isoforms. Both methods showed increased plasma TIMP-1 values for cancer patients as compared to healthy individuals. The p values for the separation of the groups were 0.0019 for ELISA and <0.0001 for SELDI TOF MS. CRC did not fundamentally affect the appearance of TIMP-1 as evaluated by SELDI TOF MS.

Rapid and Individual-specific Glycoprofiling of the Low Abundance N-Glycosylated Protein Tissue Inhibitor of Metalloproteinases-1

A gel-based method for a mass spectrometric site-specific glycoanalysis was developed using a recombinant glycoprotein expressed in two different cell lines. Hydrophilic interaction liquid chromatography at nanoscale level was used to enrich for glycopeptides prior to MS. The glycoprofiling was performed using matrix-assisted laser desorption/ionization MS and MS/MS. The method proved to be fast and sensitive and furthermore yielded a comprehensive site-specific glycan analysis, allowing a differentiation of the glycoprofiles of the two sources of recombinant protein, both comprising N-glycans of a highly heterogeneous nature. To test the potential of the method, tissue inhibitor of metalloproteinases-1 (TIMP-1), a secreted low abundance N-glycosylated protein and a cancer marker, was purified in an individual-specific manner from plasma of five healthy individuals using IgG depletion and immunoaffinity chromatography. The corresponding TIMP-1 glycoprofiles were determined to be highly similar, comprising mainly bi- and triantennary complex oligosaccharides. Additionally it was shown that platelet-derived TIMP-1 displayed a similar glycoprofile. This is the first study to investigate the glycosylation of naturally occurring human TIMP-1, and the high similarity of the glycoprofiles showed that individual-specific glycosylation variations of TIMP-1 are minimal. In addition, the results showed that TIMP-1 derived from platelets and plasma is similarly glycosylated. This comprehensive and rapid glycoprofiling of a low abundance glycoprotein performed in an individual-specific manner allows for future studies of glycosylated biomarkers for person-specific detection of altered glycosylation and may thus allow early detection and monitoring of diseases.

Investigation of tissue inhibitor of metalloproteinases 1 in plasma from colorectal cancer patients and blood donors by surface-enhanced laser desorption/ionization time-of-flight mass spectrometryscopy

Early detection of colorectal cancer (CRC) improves patient survival. Plasma tissue inhibitor of metalloproteinases 1 (TIMP-1) measurements by enzyme-linked immunosorbent assay (ELISA) have been suggested as a new method for the early detection of CRC. To further investigate the nature of TIMP-1 in plasma, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI TOF MS) was used. TIMP-1 measurements of plasma from 16 healthy donors and 14 CRC patients were performed using TIMP-1 monoclonal antibody in SELDI TOF MS and ELISA. SELDI TOF MS applying an antibody to TIMP-1 revealed that human plasma TIMP-1 has a mass of 25.1 kDa and exhibits several isoforms. Both methods showed increased plasma TIMP-1 values for cancer patients as compared to healthy individuals. The p values for the separation of the groups were 0.0019 for ELISA and <0.0001 for SELDI TOF MS. CRC did not fundamentally affect the appearance of TIMP-1 as evaluated by SELDI TOF MS.

Apolipoprotein E Knockout Mice Over-Expressing Human Tissue Inhibitor of Metalloproteinase 1 Are Protected against Aneurysm Formation but Not against Atherosclerotic Plaque Development

Aims: We investigated the effect of plasma levels of human tissue inhibitor of metalloproteinase (hTIMP)-1 on arterial lesion development and aneurysm formation in apolipoprotein-E-deficient mice (ApoE^–/–). Methods: Control and transgenic mice were fed either a chow diet or a high-fat diet for 90 and 180 days. Results: hTIMP-1 has a tendency to decrease atherosclerotic lesions, but did not attain significance (approximately 6% reduction in hTIMP-1^+/+, p = 0.075, and approximately 4% in hTIMP-1^+/0, p = 0.088 vs. control). Immunohistological and histological analyses revealed a reduction in macrophage accumulation (23% of control in hTIMP^+/0, p = 0.065, and 49% of control in hTIMP^+/+, p < 0.05) but not in collagen degradation within the lesion in transgenic mice. Moreover, elastin degradation in sites of pseudo-microaneurysms was reduced in transgenic mice (37% of control in hTIMP-1^+/0, p < 0.05, and 50% of control in hTIMP-1^+/+, p < 0.05). DNA array analysis of matrix metalloproteinase (MMP) expression followed by real-time PCR quantification revealed a significant up-regulation of MMP-3, MMP-12 and MMP-13 in arterial lesions of ApoE^–/– mice fed a high-fat diet in comparison with the same mice fed a chow diet. Conclusion: These data show that hTIMP-1 reduces aneurysm formation in ApoE^–/– mice but does not protect them against the development of arterial lesions.

Suppression effects of human recombinant tissue inhibitor of metalloproteinases-1(TIMP-1) on tumor proliferation using in vivo treatment model of well-differentiated colon cancer cell line, HT29.

To investigate the suppressive effect of human recombinant TIMP-1 (rh-TIMP-1) on tumor proliferation using an in vivo xenograft system, HT29 was suspended in 0.1 ml phosphate buffered saline (PBS) and then subcutaneously injected in the back of female mice (BALB/C nu/nu). The mice were divided into 2 groups an and the tumor diameter was measured after rh-TIMP-1 (2 mg/kg) (rh-TIMP-1 group) or PBS (control group) was administered injections according to the following schedules. Schedule 1 : Beginning 2 weeks after the subcutaneous injection of HT29, an intraperitoneal injection of rh-TIMP-1 or PBS were performed twice a day (every 12 h) for 14 consecutive days. Schedule 2 : Beginning 1 week after the subcutaneous injection of HT29, an intraperitoneal injection was performed twice a day for 14 consecutive days. Schedule 3 : Intraperitoneal injections were started simultaneously with the subcutaneous injection of HT29, and then performed twice a day for 21 consecutive days. The mice were sacrificed and the tumors extirpated for immunohistochemical investigation. In addition, gelatin zymography and a cell proliferation assay were performed. With Schedule 1, the changes in the tumor diameter in the rh-TIMP-1 group followed the same course as those in the control group, and no suppressive effect on tumor proliferation was observed. However, with Schedule 3, a remarkable suppressive effect was observed throughout the treatment period. In immunostaining, more cases negative for MMP-9 were observed in the rh-TIMP-1 group than in the control group. Cases negative for CD34 were significantly more observed in the rh-TIMP-1 group than in the control group with Schedule 3. All of the results were obtained through the suppressive effect of rh-TIMP-1 on angiogenesis.