Human MMP-9 Research Articles

Abstract 529: Atorvastatin and Sildenafil Reduce Vascular Remodeling and Oxidative Stress in 2K1C-hypertension

Atorvastatin (ATORVA) and sildenafil (SILD) exert beneficial effects on cardiovascular diseases through their pleiotropic effects. Hypertension-induced vascular hypertrophy is associated with oxidative stress and matrix metalloproteinase (MMP) up-regulation. We evaluated the effects of ATORVA and SILD on vascular changes induced by MMPs and on oxidative stress and MMP activity in 2 kidney-1 clip (2K1C) hypertension. Sham and 2K1C rats were treated with vehicle, ATORVA (50 mg/kg), SILD (45 mg/kg) or both for 8 weeks. ATORVA, SILD, or both drugs exerted antihypertensive effects (systolic blood pressure: 148±6, 156±2, and 138±4 mmHg, respectively, vs. 200±4 mmHg in 2K1C untreated rats; P<0.05). All treatments prevented the increases in the aortic cross-sectional area and media/lumen ratio in 2K1C rats (P<0.05). Aortas from 2K1C rats showed higher MMP-2 levels when compared with sham and all treatments attenuated 2K1C hypertension-induced increases in MMP-2 levels (both P<0.05). Increased gelatinolytic activity (20.6±0.9 vs. 13.9±1.2 A.U.; arbitrary units) co-localized with upregulated aortic MMP-2 expression (8.1±0.3 vs. 5.4±0.4 A.U.) in 2K1C vs. control rats. ATORVA, SILD, or both drugs lowered gelatinolytic activity to 14.6±0.5, 14.6±1.0 and 14.5±0.8 A.U. and lowered the aortic MMP-2 expression to 5.8±0.3, 5.9±0.5 and 5.5±0.5 A.U., respectively. However, these drugs had no in vitro effects on human recombinant MMP-2 activity (P>0.05). Hypertension induced oxidative stress assessed with dihydroethidium probe (7.9±1.2 vs. 16.5±1.5 A.U. in sham vs 2K1C), which was attenuated by ATORVA, SILD or both (9.9±0.3, 10.4±1.5 and 9.6±0.6 A.U., respectively). Plasma malondialdehyde levels paralleled dihydroethidium results. Treatment with ATORVA or SILD, or both, exert antihypertensive effects and prevents 2K1C hypertension-induced vascular remodeling and oxidative stress. These effects were associated with lower MMP-2 levels possibly resulting of antioxidants effects. Our results suggest that ATORVA and SILD may prevent the vascular alterations of hypertension.Supported by FAPESP, Cnpq.

Development and validation of novel enzyme activity methods to assess inhibition of matrix metalloproteinases (MMPs) in human serum by antibodies against enzyme therapeutics

This paper summarizes the development and validation of five enzyme activity methods to assess the specific inhibition of human endogenous matrix metalloproteinases MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), MMP-8 (collagenase 2) and MMP-13 (collagenase 3) by anti-Collagenase Clostridium histolyticum (CCH) antibodies in human serum. These MMPs are of interest since antibodies against a therapeutic enzyme may cross-react with, and inactivate, the MMPs. The validated methods utilize spiked exogenous individual MMPs added to serum to determine if the serum inhibits MMP enzyme activity. Factors evaluated and optimized during development include pH, reaction time and temperature, inhibitor concentration for the positive control, and substrate and serum concentration. Characteristics established during validation for each MMP activity inhibition method included intra- and inter-assay precision and recovery, recovery in the pooled normal human serum samples, bench-top stability at room temperature and on wet ice, and assay cut-point determination. Precision results ranged from ~1 to 12% CV, recoveries of the activities of the exogenous MMPs ranged from ~84 to 90% and cut-point values ranged from 67 to 91%.

P-0029 Clinicopathological Significance and Expression of KiSS-1 and MMP-9 in Gastric Carcinoma and the Precancerous Lesions

ABSTRACT Introduction To explore the relationship between KiSS-1 and MMP-9 expression in gastric carcinomas (GC); to analyze the correlation between KiSS-1 and MMP-9 expression in gastric carcinoma and relations with clinicopathological features and clinical outcomes of patients. Methods Clinical materials: 164 cases of surgically resected gastric carcinoma (GC) specimens, 164 normal gastric mucosa, 42 chronic atrophic gastritis (CAG), 93 intestinal metaplasia (IM) and 15 dysplasia (Dys) were collected from the First Affiliated Hospital of China Medical University. Immunohistochemical method was used, with mouse monoclonal antibodies against human KiSS-1 (working dilution 1:300) and MMP-9, to detect the expression of KiSS-1 and MMP-9 in normal gastric mucosa, IM, Dys and GCs. Immunohistochemical staining results assessment: Specific immunoreactivity of KISS-1 was located in the nucleus of carcinoma cells. Specific immunoreactivity of MMP-9 was located in the cytoplasm of carcinoma cells. Grading of immunostaining results: 200 cells from two selected representative fields of each sample were counted by two independent observers. The positive expression of KiSS-1 in the normal gastric mucosa as a benchmark, the expression of KiSS-1 in GCs is divided into four groups: full-negative group A, the expression of normal gastric mucosa and GC were negative, 22 cases; no changes group B, the expression of normal gastric mucosa and GC showed no significant, 59 cases; down regulation group C, the expression in GC was lower than paired normal gastric mucosa,55 cases; up regulation group D, the expression in GC was higher than paired normal gastric mucosa, 26 cases. Results The positivity rate of KiSS-1 expression was significantly lower in GCs (67.90%, 110/162) than in normal gastric (83.59%, 136/162), CAG (90.04%, 38/42) and IM (83.87%, 78/93), P 0.05). In intestinal type gastric cancer (90.48%, 38/42) the positive expression was significantly higher the diffuse-type gastric cancer (74.11%, 83/112) (P 0.05). Conclusion Decreased expression of KiSS-1 and increased expression of MMP-9 exist in GCs, and is related to certain clinicopathological parameters, suggesting they play important roles in gastric carcinogenesis. The further study is needed to explore the relevant molecular mechanism.

Opticin production is reduced by hypoxia and VEGF in human retinal pigment epithelium via MMP-2 activation

Opticin, a small leucine rich repeat protein (SLRP) contributes to vitreoretinal adhesion. This study was conducted to investigate the effects of hypoxia and vascular endothelial growth factor (VEGF) on matrix metalloproteinase (MMP) mediated opticin production in retinal pigment epithelium (RPE) cells. Primary cultured human RPE cells were treated with hypoxia (low oxygen and cobalt chloride) or VEGF (0–100ng/mL). The mRNA levels of opticin and the protein levels of intra and extracellular opticin in RPE cells were examined by RT-PCR and Western blot assay, respectively. Furthermore, the MMP activity was analyzed by zymography, and EDTA was used as an MMP inhibitor. Analysis of the effect of MMP-2 on opticin was performed by recombinant human (rh) MMP-2 stimulation in RPE cultures and by human vitreous sample digestion with activated rhMMP-2. Our results showed that opticin was expressed by primary cultured human RPE cells. Hypoxia and VEGF stimulation did not alter opticin mRNA and protein expression in RPE cells, but markedly decreased the protein levels of extracellular opticin following increased latent MMP-2 activity. The VEGF- and hypoxia induced opticin degradation in the culture medium was blocked by EDTA. Together, opticin levels in the culture medium were also reduced after rhMMP-2 treatment. In addition, opticin in human vitreous samples could be cleaved by rhMMP-2. These results reveal that VEGF and hypoxia could decrease opticin protein levels in the human RPE secretome, and that opticin may be an enzymatic substrate for MMP-2.

Abstract 1991: Effects of isoliquiritigenin (ISL) on VEGF secretion in Human breast cancer cell line MDA-MB-231

Abstract Breast cancer is the most frequent cause of mortality among females in the world. The death reasons of most breast cancer patients were due to the tumor metastasis from breast to other organs. The principal mechanisms involved in metastasis are migration and invasion. Unfortunately, there are almost no efficacious therapeutic modalities. Thus, it is imperative to develop novel targets and agents to prevent invasion and metastasis of breast cancer cells.This purpose of this study is to investigate the anti-metastatic potency of ISL via modulation of the expression of VEGF and MMP in the highly metastatic human breast cancer cell line MDA-MB-231. Based on ELISA and western blot analysis, ISL treatment (0.1-10 μM) for 24 h and 48 h reduced secretions and protein levels of VEGF in a time- and dose-dependent manner. ISL decreased the hypoxia-inducible factor 1-alpha (HIF-1α) protein expression, which is the up-regulator of VEGF. According to chamber migration assay and wound migration assay, ISL significantly suppressed the migration of MDA-MB-231 cells at 24 h and 48 h. Among human MMPs, MMP-2 and MMP-9 have been of particular interest because they efficiently cleave the major components of ECM. As confirmed by western blot and gelatin zymography assay, ISL inhibited the protein expression and gelatinolytic activity of MMP-2/-9 at 48 h. To further clarify the signal pathway responsible for ISL-downregulated cell migration in MDA-MB-231 cells, we examine the effect of ISL on PI3K/Akt pathway and MAPK pathway, as well as the NF-κB. The expression of PI3K and phosphorylation of p38 mitogen-activated protein kinase and Akt kinase were suppressed after ISL treatment. In addition, NF-κB DNA binding assay demonstrated that NF-κB DNA binding activity was inhibited after ISL treatment. Taken together, these findings suggest the ISL inhibits p38, PI3K/Akt, and NF-κB signaling, resulting in the inhibition of VEGF, HIF-1α, MMP-9, and MMP-2, and thus suppresses migration of MDA-MB-231 cells. We believe that these findings will provide the potential application of ISL on the treatment of metastatic breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1991. doi:1538-7445.AM2012-1991

Cartilage Destruction in Granulomatosis with Polyangiitis (Wegener's Granulomatosis) Is Mediated by Human Fibroblasts after Transplantation into Immunodeficient Mice

A key feature of granulomatosis with polyangiitis (GPA; or Wegener's granulomatosis) is the granulomatous inflammation of the upper respiratory tract, which leads to the subsequent destruction of adjacent tissues. The aim of our work was to study the histopathological and cellular components of tissue destruction of human GPA tissue transplanted into immunodeficient mice. Biopsy specimens from patients with active GPA (n = 10) or sinusitis (controls, n = 6) were s.c. co-implanted with healthy allogeneic human nasal cartilage into immunodeficient pfp/rag2(-/-) mice. Transplants were examined for their destructive capability of the allografted human cartilage. In addition, nasal fibroblasts from patients with GPA (n = 8) and control healthy nasal fibroblasts (n = 5) were cultured, and cell proliferation and apoptosis were quantified. mRNA and protein levels of matrix metalloproteinases and cytokines were evaluated at baseline and after proinflammatory stimulation. GPA implants showed massive destruction of the co-implanted human cartilage, whereas cartilage destruction was only marginal in control samples. Destruction was mediated by human fibroblasts and could be inhibited by corticoid treatment. The up-regulated production of matrix metalloproteinases 1, 3, and 13 and cytokines IL-6 and IL-8 was found in vivo and in vitro. Although proliferation of isolated fibroblasts was comparable between GPA and controls, GPA samples showed a significant delay of apoptosis. The destruction of nasal cartilage in GPA is mainly mediated by fibroblasts that can be blocked by corticosteroids, and this tissue destruction is not dependent on the influx of leukocytes.

Design of Barbiturate–Nitrate Hybrids that Inhibit MMP-9 Activity and Secretion

We describe a new type of barbiturate-based matrix metalloproteinase (MMP) inhibitor incorporating a nitric oxide (NO) donor/mimetic group (series 1). The compounds were designed to inhibit MMP at enzyme level and to attenuate MMP-9 secretion arising from inflammatory signaling. To detect effects related to the nitrate, we prepared and studied an analogous series of barbiturate C5-alkyl alcohols that were unable to release NO (series 2). Both series inhibited recombinant human MMP-2/9 activity with nanomolar potency. Series 1 consistently inhibited the secretion of MMP-9 from TNFα/IL1β stimulated Caco-2 cells at 10 μM, which could be attributed to NO related effects because the non-nitrate panel did not affect enzyme levels. Several compounds from series 1 (10 μM) inhibited tumor cell invasion but none from the non-nitrate panel did. The work shows that MMP-inhibitory barbiturates are suitable scaffolds for hybrid design, targeting additional facets of MMP pathophysiology, with potential to improve risk-benefit ratios.

A smallest 6 kda metalloprotease, mini-matrilysin, in living world: a revolutionary conserved zinc-dependent proteolytic domain- helix-loop-helix catalytic zinc binding domain (ZBD)

BackgroundThe Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded β sheet and three long α helices (A, B and C). The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?”MethodsWe have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded β sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolytic activities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD.ResultsThis minimal 6 kDa matrilysin has been refolded and shown to have proteolytic activity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolytic activity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolytic activities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6 ~ 7 kDa fragments. Thus, many of the functions of the enzyme are retained indicating that the helix B-Met loop-helix C is the minimal functional “domain” found to date for the matrixin family.ConclusionsThe helix B-Met loop-helix C folding conserved in metalloprotease metzincin super family is able to facilitate proteolytic catalysis for specific substrate and inhibitor recognition. The autolysis processing and producing 6 kDa mini MMP-7 is the smallest metalloprotease in living world.

Ubiquitin-intein and SUMO2-intein fusion systems for enhanced protein production and purification

Although most commonly used for protein production, expression of soluble and functional recombinant protein in Escherichia coli is still a major challenge. The development and application of fusion tags that can facilitate protein expression and solubility partly solve this problem, however, under most circumstance, the fusion tags have to be removed by proteases in order to use the proteins. Because the tag removal using proteases increases cost and introduces extra purification steps, it remains a significant problem that must be resolved before being widely used in industry production. Ubiquitin and SUMO have been successfully used to enhance protein expression and solubility. In the last decades, intein has also been widely used in protein production for its self-cleavage property, which could help to remove the fusion tag without any protease. Here, we take the advantages of ubiquitin, SUMO2 and intein in protein expression. We constructed tandem ubiquitin-intein and SUMO2-intein fusion tags, and chose human MMP13 (amino acid 104–274) and eGFP as the passenger proteins that fused to the C-terminus of the tags. These constructs were expressed in E. coli and both MMP13 and eGFP expression and solubility were evaluated. Both tags showed the ability to enhance the solubility of MMP13 and eGFP and improve the expression of eGFP, and the SUMO2-intein having a more significant effect. Both ubiquitin-intein-eGFP and SUMO2-intein-eGFP were purified using Ni–NTA column chromatography and self-cleavaged by changing pH. The recombinant un-tagged eGFP were released and eluted with high homogeneity. In summary, ubiquitin-intein and SUMO2-intein are convenient and useful fusion tags that can enhance the expression, solubility and improve the purification process of the model heterologous protein and these tags may have a good prospect in protein production.

Abstract 9436: Sitagliptin Reduces Plaque Macrophage Content and Stabilizes Arteriosclerotic Lesions in Apoe-Deficient Mice

Objective: Glucagon-like peptide-1(7-36) (GLP-1) based therapies are current treatment options for patients with type 2 diabetes mellitus. As patients with diabetes are at a high risk to develop a severe atherosclerosis, we investigated the effect of GLP-1 on chemokine-induced migration of human monocytes and macrophage MMP-9 expression, both crucial steps in atherogenesis and plaque destabilisation. Furthermore, we explored the translation of these effects to plaque inflammation and plaque stability in ApoE -/- mice treated with the DPP-IV inhibitor sitagliptin, which increases plasma levels of active GLP-1. Methods and Results: In the modified Boyden-chamber, pretreatment with GLP-1 reduced the MCP-1 induced migration of isolated human monocytes in a concentration dependent manner from a 3.3±0.8 to a 1.4±0.5 fold induction at 10 nM GLP-1 (n = 8, p < 0.05), which was associated with a decreased MLC-phosphorylation in these cells. Similar effects were seen when RANTES was used as a chemokine to induce cell migration. Furthermore, GLP-1 significantly decreased MMP-9 release from isolated human monocyte-derived macrophages as determined by zymography. In vivo , treatment of ApoE -/- mice with the DPP-IV inhibitor sitagliptin, which increases plasma levels of active GLP-1, resulted in a 75% reduction of macrophage infiltration, a 75% reduction in plaque MMP-9 expression and a more than 3-fold increase in plaque collagen content compared to controls as shown by MAC3, Picrosirus Red and immunohistologic staining, respectively (n=6-8, p < 0.05). Conclusion: Thus, GLP-1 reduces plaque inflammation and increases plaque stability potentially by inhibiting chemokine-induced migration of monocytes and by inhibition of macrophage MMP-9 expression. The effects observed may provide a potential mechanism of how GLP-1 based therapies may modulate vascular disease in high-risk patients with type 2 diabetes mellitus.

C/EBPβ and RUNX2 cooperate to degrade cartilage with MMP-13 as the target and HIF-2α as the inducer in chondrocytes

To elucidate the molecular mechanism underlying the endochondral ossification process during the skeletal growth and osteoarthritis (OA) development, we examined the signal network around CCAAT/enhancer-binding protein-β (C/EBPβ, encoded by CEBPB), a potent regulator of this process. Computational predictions and a C/EBP motif-reporter assay identified RUNX2 as the most potent transcriptional partner of C/EBPβ in chondrocytes. C/EBPβ and RUNX2 were induced and co-localized in highly differentiated chondrocytes during the skeletal growth and OA development of mice and humans. The compound knockout of Cebpb and Runx2 in mice caused growth retardation and resistance to OA with decreases in cartilage degradation and matrix metalloproteinase-13 (Mmp-13) expression. C/EBPβ and RUNX2 cooperatively enhanced promoter activity of MMP13 through specific binding to a C/EBP-binding motif and an osteoblast-specific cis-acting element 2 motif as a protein complex. Human genetic studies failed to show the association of human CEBPB gene polymorphisms with knee OA, nor was there a genetic variation around the identified responsive region in the human MMP13 promoter. However, hypoxia-inducible factor-2α (HIF-2α), a functional and genetic regulator of knee OA through promoting endochondral ossification, was identified as a potent and functional inducer of C/EBPβ expression in chondrocytes by the CEBPB promoter assay. Hence, C/EBPβ and RUNX2, with MMP-13 as the target and HIF-2α as the inducer, control cartilage degradation. This molecular network in chondrocytes may represent a therapeutic target for OA.

Serum from Patients Undergoing Remote Ischemic Preconditioning Protects Cultured Human Intestinal Cells from Hypoxia-Induced Damage: Involvement of Matrixmetalloproteinase-2 and -9

Remote ischemic preconditioning (RIPC) can be induced by transient occlusion of blood flow to a limb with a blood pressure cuff and exerts multiorgan protection from ischemia/reperfusion injury. Ischemia/reperfusion injury in the intestinal tract leads to intestinal barrier dysfunction and can result in multiple organ failure. Here we used an intestinal cell line (CaCo-2) to evaluate the effects of RIPC-conditioned patient sera on hypoxia-induced cell damage in vitro and to identify serum factors that mediate RIPC effects. Patient sera (n = 10) derived before RIPC (T0), directly after RIPC (T1) and 1 h after RIPC (T2) were added to the culture medium at the onset of hypoxia until 48 h after hypoxia. Reverse transcription-polymerase chain reaction, lactate dehydrogenase (LDH) assays, caspase-3/7 assays, silver staining, gelatin zymography and Western blotting were performed. Hypoxia led to morphological signs of cell damage and increased the release of LDH in cultures containing sera T0 (P < 0.01) and T1 (P < 0.05), but not sera T2, which reduced the hypoxia-mediated LDH release compared with sera T0 (P < 0.05). Gelatin zymography revealed a significant reduction of activities of the matrixmetalloproteinase (MMP)-2 and MMP-9 in the protective sera T2 compared with the nonprotective sera T0 (MMP-2: P < 0.01; MMP-9: P < 0.05). Addition of human recombinant MMP-2 and MMP-9 to MMP-deficient culture media increased the sensitivity of CaCo-2 cells to hypoxia-induced cell damage (P < 0.05), but did not result in a reduced phosphorylation of prosurvival kinases p42/44 and protein kinase B (Akt) or increased activity of caspase-3/7. Our results suggest MMP-2 and MMP-9 as currently unknown humoral factors that may be involved in RIPC-mediated cytoprotection in the intestine.

Novel Yeast Bioassay for High-Throughput Screening of Matrix Metalloproteinase Inhibitors

Diverse malfunctions in the expression and regulation of matrix metalloproteinases (MMPs) are often the cause of severe human diseases, bringing the identification of specific MMP inhibitors into major focus, particularly in anticancer treatment. Here, we describe a novel bioassay based on recombinant yeast cells (Pichia pastoris) that express, deliver, and incorporate biologically active human MMP-2 and MMP-9 at the yeast cell surface. Using Sed1p for cell wall targeting and covalent anchorage, a highly efficient bioassay was established that allows high-throughput screening and subsequent validation of novel MMP inhibitors as potential anticancer drugs. In addition, we developed a straightforward synthesis of a new aspartate-derived MMP inhibitor active in the nM range and bearing an amino functionality that should allow the introduction of a wide range of side chains to modify the properties of these compounds.

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.

Potent and selective 2-naphthylsulfonamide substituted hydroxamic acid inhibitors of matrix metalloproteinase-13

The matrix metalloproteinase enzyme MMP-13 plays a key role in the degradation of type II collagen in cartilage and bone in osteoarthritis (OA). An effective MMP-13 inhibitor would provide a disease modifying therapy for the treatment of arthritis, although this goal still continues to elude the pharmaceutical industry due to issues with safety. Our efforts have resulted in the discovery of a series of hydroxamic acid inhibitors of MMP-13 that do not significantly inhibit MMP-2 (gelatinase-1). MMP-2 has been implicated in the musculoskeletal side effects resulting from pan-MMP inhibition due to findings from spontaneously occurring human MMP-2 deletions. Analysis of the SAR of hundreds of previously prepared hydroxamate based MMP inhibitors lead us to 2-naphthylsulfonamide substituted hydroxamates which exhibited modest selectivity for MMP-13 versus MMP-2. This Letter describes the lead optimization of 1 and identification of inhibitors exhibiting >100-fold selectivity for MMP-13 over MMP-2.

Matrix Metalloproteinase-13 Promotes Recovery from Experimental Liver Cirrhosis in Rats

Objective: To evaluate the role of matrix metalloproteinase (MMP)-13 gene expression in the early phase of recovery from liver fibrosis/cirrhosis. Methods: Liver fibrosis was induced in male Wistar rats by administration of carbon tetrachloride (CCl₄) for 10 weeks. Recombinant adenovirus-mediated human MMP-13 gene transfer (RAdMMP-13) was performed via the femoral vein on day 3 after the last CCl₄ injection. The role of MMP-13 in stably expressing cell lines was also analyzed. Results: Fibrous deposition in the liver was decreased in RAdMMP-13-injected rats by day 3 after gene transfer compared with empty vector RAd66-injected rats. Furthermore, MMP-2 and MMP-9 enzymatic activity was markedly enhanced in the liver of RAdMMP-13 injected rats. Hepatocyte growth factor (HGF) induction was also increased in RAdMMP-13 injected rats. In established stable HT-1080 cells transfected with MMP-13, HGF-α expression and MMP-2 and MMP-9 enzymatic activity were increased. The conversion of precursor HGF into mature HGF was also increased in the MMP-13 expressing cell lines. Conclusion: Forced MMP-13 expression effectively accelerated recovery from liver cirrhosis via the effects of MMP-13-mediated HGF, MMP-2, and MMP-9 expression, which induced the degradation of collagen fibers and promoted hepatic regeneration.

MMP-9 activity is increased by adiponectin in primary human hepatocytes but even negatively correlates with serum adiponectin in a rodent model of non-alcoholic steatohepatitis

Adiponectin protects from inflammation and fibrosis in metabolic liver disease. In the present study we analyzed whether this adipokine may directly affect the activity of matrix metalloproteinases (MMPs), central regulators of fibrinolysis, in hepatocytes. Global gene expression analysis indicated upregulation of MMP-9 and tissue inhibitor of metalloproteinases-1 (TIMP-1) expression in primary human hepatocytes (PHH) in response to stimulation with adiponectin, and these results were confirmed by real-time RT-PCR. Furthermore, gelatin zymography revealed that MMP-9 activity was significantly induced in supernatants of adiponectin stimulated PHHs. In a murine model of hepatic steatosis and in human steatotic liver samples hepatic MMP-9 activity was not significantly altered. However, in two different murine models of non-alcoholic steatohepatitis (NASH) MMP-9 activity was significantly elevated compared to chow fed control mice. Of note, MMP-9 activity did not or even negatively, respectively, correlate with adiponectin serum levels in these models. The current data indicate that in NASH hepatic inflammation and fibrosis but not hepatic steatosis induce liver MMP-9 activity, and this induction seems to be related to the anti-inflammatory activity of adiponectin rather than its effect on hepatocellular MMP-9 expression.

Localization of Matrix Metalloproteinase (MMP)-9 in Lung Tissue of a Murine Model of Allergic Asthma

MMP-9 (gelatinase B) is recognized in chronic obstructive pulmonary disease (COPD) and now asthma as playing a central role in matrix degradation in injury, as well as contributing to the remodeling process. The increasing focus on MMP-9 in human and animal research supports the need for a reliable immunostain in lung tissue. However, MMP-9 immunostaining in murine systems is hampered by several factors. First, many of the anti-human antibodies do not readily cross-react with murine MMP-9 despite the high degree of conservation between human and murine MMP-9. Secondly, the availability of detailed protocols is limited. Lung MMP-9 immunostaining is further complicated by technical issues such as edge effect, availability of positive and negative controls, antigen retrieval, staining specificity, and the need to achieve a delicate balance of primary and secondary antibody concentrations, and colorimetric reagents which will allow visualization of specific cell expression in highly delicate lung tissue, while also demonstrating adequate uptake in (extra-pulmonary) tissue controls. We describe a detailed method for immunostaining MMP-9 in mouse lung paraffin-embedded tissue utilizing human ovary as a control since MMP-9 is known to be over-expressed in human ovarian carcinomas.

A matrix metalloprotease‐PAR1 system regulates vascular integrity, systemic inflammation and death in sepsis

Sepsis is a deadly disease characterized by the inability to regulate the inflammatory–coagulation response in which the endothelium plays a key role. The cause of this perturbation remains poorly understood and has hampered the development of effective therapeutics. Matrix metalloproteases (MMPs) are involved in the host response to pathogens, but can also cause uncontrolled tissue damage and contribute to mortality. We found that human sepsis patients had markedly elevated plasma proMMP-1 and active MMP-1 levels, which correlated with death at 7 and 28 days after diagnosis. Likewise, septic mice had increased plasma levels of the MMP-1 ortholog, MMP-1a. We identified mouse MMP-1a as an agonist of protease-activated receptor-1 (PAR1) on endothelial cells. MMP-1a was released from endothelial cells in septic mice. Blockade of MMP-1 activity suppressed endothelial barrier disruption, disseminated intravascular coagulation (DIC), lung vascular permeability as well as the cytokine storm and improved survival, which was lost in PAR1-deficient mice. Infusion of human MMP-1 increased lung vascular permeability in normal wild-type mice but not in PAR1-deficient mice. These findings implicate MMP-1 as an important activator of PAR1 in sepsis and suggest that therapeutics that target MMP1-PAR1 may prove beneficial in the treatment of sepsis.

MMP-1 drives immunopathology in human tuberculosis and transgenic mice

Mycobacterium tuberculosis can cause lung tissue damage to spread, but the mechanisms driving this immunopathology are poorly understood. The breakdown of lung matrix involves MMPs, which have a unique ability to degrade fibrillar collagens at neutral pH. To determine whether MMPs play a role in the immunopathology of tuberculosis (TB), we profiled MMPs and their inhibitors, the tissue inhibitor of metalloproteinases (TIMPs), in sputum and bronchoalveolar lavage fluid from patients with TB and symptomatic controls. MMP-1 concentrations were significantly increased in both HIV-negative and HIV-positive patients with TB, while TIMP concentrations were lower in HIV-negative TB patients. In primary human monocytes, M. tuberculosis infection selectively upregulated MMP1 gene expression and secretion, and Ro32-3555, a specific MMP inhibitor, suppressed M. tuberculosis-driven MMP-1 activity. Since the mouse MMP-1 ortholog is not expressed in the lung and mice infected with M. tuberculosis do not develop tissue destruction equivalent to humans, we infected transgenic mice expressing human MMP-1 with M. tuberculosis to investigate whether MMP-1 caused lung immunopathology. In the MMP-1 transgenic mice, M. tuberculosis infection increased MMP-1 expression, resulting in alveolar destruction in lung granulomas and significantly greater collagen breakdown. In summary, MMP-1 may drive tissue destruction in TB and represents a therapeutic target to limit immunopathology.