Engineering metalloproteinase inhibitors: tissue inhibitors of metalloproteinases or antibodies, that is the question.

Targeting metalloproteinases (MPs) has been the center of attention for developing therapeutics due to their contribution to a wide range of diseases, including cancer, cardiovascular, neurodegenerative disease, and preterm labor. Protein-based MP inhibitors offer higher stability and selectivity, which is critical for developing efficient therapeutics with low off-target effects. Tissue inhibitors of metalloproteinases (TIMPs), natural inhibitors of MPs, and antibodies provide excellent protein scaffolds for engineering selective or multispecific MP inhibitors. Advances in protein engineering and design techniques, such as rational design and directed evolution using yeast display to develop potent MP inhibitors, are discussed, including but not limited to loop grafting, swapping, and counterselective selection.

Serum Amyloid P Attenuates Hypertrophic Scarring in Large Animal Models.

INTRODUCTION: This study's purpose was to (1)determine the effect of locally administered serum amyloid P (SAP) on the development of hypertrophic scars (HTS) in porcine and rabbit HTS models and (2)determine the pharmacokinetics of systemically administered SAP and its effect on circulating fibrocyte quantities. METHODS: Two large animal (New Zealand White Rabbit and Female Red Duroc Pigs) HTS models were utilized to study the effects of daily local injections of SAP immediately post wounding (x5 d in rabbits; x7 d in pigs) on HTS development as measured by scar elevation index , scar area, wound closure, and molecular expression studies of scar components. For SAP pharmacokinetics, total and human SAP levels in porcine blood were measured at regular intervals following intravenous administration of human SAP. Fibrocyte quantities were determined prior to and 1 h following human SAP intravenous administration. RESULTS: In the rabbit model, local SAP significantly decreased the level of tissue inhibitor of metalloproteinases-1 mRNA expression and maintained matrix mettaloproteinase-9 expression, while control and vehicle groups significantly declined. In the pig model, there was a significant decrease in the trend of scar elevation indexes treated with local SAP versus controls over the study period. This decrease was statistically significant at days 14 and 84. Human SAP administered intravenously is degraded within 24 h and does not influence circulating fibrocyte quantities. CONCLUSIONS: This is the first study to demonstrate attenuation of HTS formation using locally administered SAP in large animal HTS models. Local SAP administration reduces HTS formation by maintaining matrix mettaloproteinase-9 and decreasing tissue inhibitor of metalloproteinases-1. Intravenous administration of SAP is not as effective.

Mechanism and Prevention of Atrial Remodeling and Their Related Genes in Cardiovascular Disorders.

Atrial fibrillation (AF) is associated with profound structural and functional changes in the atrium. Inflammation mediated atrial fibrosis is one of the key mechanisms in the pathogenesis of AF. The collagen deposition in extracellular matrix (ECM) is mainly mediated by transforming growth factor ß1 (TGF-ß1) which promotes AF via controlling smads mediated-collagen gene transcription and regulating the balance of metalloproteinases (MMPs)/ tissue inhibitor of metalloproteinases (TIMPs). Although many processes can alter atrial properties and promote AF, animal models and clinical studies have provided insights into 2 major forms of atrial remodeling: Atrial tachycardia remodeling (ATR), which occurs with rapid atrial tachyarrhythmia's such as AF and atrial flutter, and atrial structural remodeling (ASR), which is associated with CHF and other fibrosis-promoting conditions. The mechanism of atrial remodeling such as atrial enlargement, ultra-structural changes of atrial muscle tissue and myocardial interstitial fibrosis in AF is still unclear. At present, many studies focus on calcium overload, renin angiotensin aldosterone system and transforming growth factor ß1, that effect on atrial structural remodeling. Recent experimental studies and clinical investigations have provided structural remodeling is important contributor to the AF. This paper reviews the current understanding of the progresses about mechanism of atrial structural remodeling, and highlights the potential therapeutic approaches aimed at attenuating structural remodeling to prevent AF. Now some recent advancements of this area are reviewed in this paper.

Plantamajoside Attenuates Neointima Formation via Upregulation of Tissue Inhibitor of Metalloproteinases in Balloon-Injured Rats.

The abnormal change of vascular smooth muscle cell (VSMC) behavior is an important cellular event leading to neointimal hyperplasia in atherosclerosis and restenosis. Plantamajoside (PMS), a phenylethanoid glycoside compound of the Plantago asiatica, has been reported to have anti-inflammatory, antioxidative, and anticancer activities. In this study, the protective effects of PMS against intimal hyperplasia and the mechanisms underlying the regulation of VSMC behavior were investigated. MTT and BrdU assays were performed to evaluate the cytotoxicity and cell proliferative activity of PMS, respectively. Rat aortic VSMC migrations after treatment with the determined concentration of PMS (50 and 150 µM) were evaluated using wound healing and Boyden chamber assays. The inhibitory effects of PMS on intimal hyperplasia were evaluated in balloon-injured (BI) rat carotid artery. PMS suppressed the proliferation in platelet-derived growth factor-BB-induced VSMC, as confirmed from the decrease in cyclin-dependent kinase (CDK)-2, CDK-4, cyclin D1, and proliferating cell nuclear antigen levels. PMS also inhibited VSMC migration, consistent with the downregulated expression and zymolytic activities of matrix metalloproteinase (MMP)2, MMP9, and MMP13. PMS specifically regulated MMP expression through p38 mitogen-activated protein kinase and focal adhesion kinase pathways. Tissue inhibitor of metalloproteinase (TIMP)1 and TIMP2 levels were upregulated via Smad1. TIMPs inhibited the conversion of pro-MMPs to active MMPs. PMS significantly inhibited neointimal formation in BI rat carotid arteries. In conclusion, PMS inhibits VSMC proliferation and migration by upregulating TIMP1 and TIMP2 expression. Therefore, PMS could be a potential therapeutic agent for vascular atherosclerosis and restenosis treatment.

Modulation of peritumoral fibroblasts with a membrane-tethered tissue inhibitor of metalloproteinase (TIMP) for the elimination of cancer cells.

BACKGROUND: Peritumoral fibroblasts are key components of the tumor microenvironment. Through remodeling of the extracellular matrix (ECM) and secretion of pro-tumorigenic cytokines, peritumoral fibroblasts foster an immunosuppressive milieu conducive to tumor cell proliferation. In this study, we investigated if peritumoral fibroblasts could be therapeutically engineered to elicit an anti-cancer response by abolishing the proteolytic activities of membrane-bound metalloproteinases involved in ECM modulation. METHODS: A high affinity, glycosylphosphatidylinositol (GPI)-anchored Tissue Inhibitor of Metalloproteinase (TIMP) named "T1PrαTACE" was created for dual inhibition of MT1-MMP and TACE. T1PrαTACE was expressed in fibroblasts and its effects on cancer cell proliferation investigated in 3D co-culture models. RESULTS: T1PrαTACE abrogated the activities of MT1-MMP and TACE in host fibroblasts. As a GPI protein, T1PrαTACE could spontaneously detach from the plasma membrane of the fibroblast to co-localize with MT1-MMP and TACE on neighboring cancer cells. In a 3D co-culture model, T1PrαTACE promoted adherence between the cancer cells and surrounding fibroblasts, which led to an attenuation in tumor development. CONCLUSION: Peritumoral fibroblasts can be modulated with the TIMP for the elimination of cancer cells. As a novel anti-tumor strategy, our approach could potentially be used in combination with conventional chemo- and immunotherapies for a more effective cancer therapy.

Abiotic Mimic of Endogenous Tissue Inhibitors of Metalloproteinases: Engineering Synthetic Polymer Nanoparticles for Use as a Broad-Spectrum Metalloproteinase Inhibitor.

We describe a process for engineering a synthetic polymer nanoparticle (NP) that functions as an effective, broad-spectrum metalloproteinase inhibitor. Inhibition is achieved by incorporating three functional elements in the NP: a group that interacts with the catalytic zinc ion, functionality that enhances affinity to the substrate-binding pocket, and fine-tuning of the chemical composition of the polymer to strengthen NP affinity for the enzyme surface. The approach is validated by synthesis of a NP that sequesters and inhibits the proteolytic activity of snake venom metalloproteinases from five clinically relevant species of snakes. The mechanism of action of the NP mimics that of endogenous tissue inhibitors of metalloproteinases. The strategy provides a general design principle for synthesizing abiotic polymer inhibitors of enzymes.

Neural stem cells and the secreted proteins TIMPs ameliorate UVB-induced skin photodamage.

UV-induced skin damage is involved in ROS overproduction and the overexpression of matrix metalloproteinases (MMPs), which are inhibited by TIMPs (tissue inhibitor of neural stem cells (NSCs)). These proteins may be associated with skin regeneration through the activation of TIMP proteins, but there have been no reports of treatment of skin photodamage using NSCs and their secreted proteins TIMP-1 and TIMP-2. Here we investigated the photoprotective role of NSCs and their TIMP proteins for the inhibition of UVB-irradiation damage in fibroblasts in SKH-1 mice. SKH-1 hairless mice were divided into three groups (n = 4 per group): normal, treatment, and control groups. The latter two groups were dorsally exposed to UVB irradiation for 12 weeks. After UVB irradiation, treatments with NSC-CM and its secreted factors TIMP-1 and TIMP-2, markedly ameliorated the photodamage triggered by the increase in MMP expression and activity through ROS production, and the subsequent activation of the NF-κB pathway in UVB-irradiated fibroblasts and the treatment mouse group. In addition, the topical application of NSC-CM to mice in the treatment group after irradiation clearly inhibited the expression of γ-H2AX, a DNA damage marker, through the activation of the DNA repair enzyme Rad50. These results demonstrate that NSC-CM or TIMPs proteins can ameliorate skin photodamage induced by UVB-irradiation in in vitro and in vivo systems.

Tyrosine phosphorylation of the transmembrane protein SIRPα: Sensing synaptic activity and regulating ectodomain cleavage for synapse maturation.

Synapse maturation is a neural activity-dependent process during brain development, in which active synapses preferentially undergo maturation to establish efficient neural circuits in the brain. Defects in this process are implicated in various neuropsychiatric disorders. We have previously reported that a postsynaptic transmembrane protein, signal regulatory protein-α (SIRPα), plays an important role in activity-dependently directing synapse maturation. In the presence of synaptic activity, the ectodomain of SIRPα is cleaved and released and then acts as a retrograde signal to induce presynaptic maturation. However, how SIRPα detects synaptic activity to promote its ectodomain cleavage and synapse maturation is unknown. Here, we show that activity-dependent tyrosine phosphorylation of SIRPα is critical for SIRPα cleavage and synapse maturation. We found that during synapse maturation and in response to neural activity, SIRPα is highly phosphorylated on its tyrosine residues in the hippocampus, a structure critical for learning and memory. Tyrosine phosphorylation of SIRPα was necessary for SIRPα cleavage and presynaptic maturation, as indicated by the fact that a phosphorylation-deficient SIRPα variant underwent much less cleavage and could not drive presynaptic maturation. However, SIRPα phosphorylation did not affect its synaptic localization. Finally, we show that inhibitors of the Src and JAK kinase family suppress neural activity-dependent SIRPα phosphorylation and cleavage. Together, our results indicate that SIRPα phosphorylation serves as a mechanism for detecting synaptic activity and linking it to the ectodomain cleavage of SIRPα, which in turn drives synapse maturation in an activity-dependent manner.

Structural and functional analyses of a TIMP and MMP in the ligament of Pinctada fucata.

The bivalve hinge ligament is the hard tissue that functions to open and close shells. The ligament contains fibrous structures consisting of aragonite crystals surrounded by a dense organic matrix. This organic matrix may contribute to the formation of fibrous aragonite crystals, but the mechanism underlying this formation remains unclear. In this study, we identified a novel ligament-specific protein, Pinctada fucata tissue inhibitor of metalloproteinase (PfTIMP), from the fibrous organic matrix between aragonite crystals in the ligament using the amino acid sequence and cDNA cloning methods. PfTIMP consists of 143 amino acid residues and has a molecular weight of 13,580.4. To investigate the activity of PfTIMP, inhibition of matrix metalloproteinase (MMP) activity was measured. PfTIMP strongly inhibited human MMP13 and MMP9. Eight MMP homologs were identified from a P. fucata genomic database by BLAST search. To identify the specific MMP that may contribute to ligament formation, the expression level of each MMP was measured in the mantle isthmus, which secretes the ligament. The expression of MMP54089 increased after scratching of the ligament, while the expressions of other MMPs did not increase after doing the same operation. To identify the role of MMP54089 in forming the ligament structure, double stranded (ds) RNA targeting MMP54089 was injected into living P. fucata to suppress the function of MMP54089. Scanning electron microscopic images showed disordered growing surfaces of the ligament in individuals injected with MMP54089-specific dsRNA. These results suggest that PfTIMP and MMP54089 play important roles in the formation of the fibrous ligament structure.

High-fat diet-induced obesity regulates MMP3 to modulate depot- and sex-dependent adipose expansion in C57BL/6J mice.

Increased adipocyte size is hypothesized to signal the recruitment of adipose progenitor cells (APCs) to expand tissue storage capacity. To investigate depot and sex differences in adipose growth, male and female C57BL/6J mice (10 wk-old) were challenged with high-fat (HF) or low-fat (LF) diets (D) for 14 wk. The HFD increased gonadal (GON) depot weight by adipocyte hypertrophy and hyperplasia in females but hypertrophy alone in males. In both sexes, inguinal (ING) adipocytes were smaller than GON, and depot expansion was due to hypertrophy. Matrix metalloproteinase 3 (Mmp3), an antiadipogenic factor, and its inhibitor Timps modulate the extracellular matrix remodeling needed for depot expansion. Mmp3 mRNA was depot different (ING > GON), higher in females than males and mainly expressed in APCs. In males, HFD-induced obesity increased tissue and APC Mmp3 mRNA levels and MMP3 protein and enzymatic activity. In females however, HFD significantly decreased MMP3 protein without affecting its mRNA levels. MMP3 activity also decreased (significant in ING). Timp4 mRNA was expressed mainly in adipocytes, and HFD-induced obesity tended to increase the ratio of TIMP4 to MMP3 protein in females, whereas it decreased it in males. Overexpression of Mmp3 in 3T3-L1 preadipocytes or rhMMP3 protein added to primary human preadipocytes inhibited differentiation, whereas rhTIMP4 improved adipogenesis and attenuated the inhibitory effect of rhMMP3. These data suggest that HFD-induced obesity downregulates APC MMP3 expression to trigger adipogenesis, and adipocyte TIMP4 may modulate this process to regulate hyperplastic vs. hypertrophic adipose tissue expansion, fat distribution, and metabolic health in a sex- and depot-dependent manner.

TIMP4 Modulates ER-α Signalling in MCF7 Breast Cancer Cells.

Tissue inhibitor of metalloprotease 4 (TIMP4) contributes to poor prognosis in breast and other tumours. However, the mechanisms of how TIMP4 influences breast cancer cell behaviour are unknown. Our aim was to explore the signalling pathways modulated by TIMP4 in breast cancer cells. Human recombinant TIMP4 was added to MCF7 breast cancer cells and RNASeq was performed. TIMP4 RNASeq results were validated by RT-PCR. Network analyses of TIMP4-exposed cells showed that ER-α, HIF1A and TGF-ß signalling were activated, whereas FOXO3 signalling was downregulated. ER-α protein levels were increased and concordantly, promoters of TIMP4-upregulated genes were significantly enriched in oestrogen-binding sites. We concluded that TIMP4 modulates multiple signalling pathways relevant in cancer in MCF7 cells, including the ER-α cascade.

Expanding the Activity of Tissue Inhibitors of Metalloproteinase (TIMP)-1 against Surface-Anchored Metalloproteinases by the Replacement of Its C-Terminal Domain: Implications for Anti-Cancer Effects.

Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous inhibitors of the matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs). TIMP molecules are made up of two domains: an N-terminal domain that associates with the catalytic cleft of the metalloproteinases (MP) and a smaller C-terminal domain whose role in MP association is still poorly understood. This work is aimed at investigating the role of the C-terminal domain in MP selectivity. In this study, we replaced the C-terminal domain of TIMP-1 with those of TIMP-2, -3 and -4 to create a series of "T1:TX" chimeras. The affinity of the chimeras against ADAM10, ADAM17, MMP14 and MMP19 was investigated. We can show that replacement of the C-terminal domain by those of other TIMPs dramatically increased the affinity of TIMP-1 for some MPs. Furthermore, the chimeras were able to suppress TNF-α and HB-EGF shedding in cell-based setting. Unlike TIMP-1, T1:TX chimeras had no growth-promoting activity. Instead, the chimeras were able to inhibit cell migration and development in several cancer cell lines. Our findings have broadened the prospect of TIMPs as cancer therapeutics. The approach could form the basis of a new strategy for future TIMP engineering.

Pharmacological strategies for protection of extrahepatic islet transplantation.

The safety and effectiveness of islet transplantation has been proven through world-wide trials. However, acute and chronic islet loss has hindered the ultimate objective of becoming a widely used treatment option for type 1 diabetes. A large islet loss is attributed, in part, to the liver being a less-than-optimal site for transplantation. Over half of the transplanted islets are destroyed shortly after transplantation due to direct exposure to blood and non-specific inflammation. Successfully engrafted islets are continuously exposed to the liver micro-environment, a unique immune system, low oxygen tension, toxins and high glucose, which is toxic to islets, leading to premature islet dysfunction/death. Investigations have continued to search for alternate sites to transplant islets that provide a better environment for prolonged function and survival. This article gathers courses and conditions that lead to islet loss, from organ procurement through islet transplantation, with special emphasis on hypoxia, oxidative stress, and antigen non-specific inflammation, and reviews strategies using pharmacological agents that have shown effectiveness in protecting islets, including a new treatment approach utilizing siRNA. Pharmacological agents that support islet survival and promote ß-cell proliferation are also included. Treatment of donor pancreata and/or islets with these agents should increase the effectiveness of islets transplanted into extrahepatic sites. Furthermore, the development of methods designed to release these agents over an extended period, will further increase their efficacy. This requires the combined efforts of both islet transplant biologists and bioengineers.

Pigment epithelium-derived factor as a natural matrix metalloproteinase inhibitor: a comparison with classical matrix metalloproteinase inhibitors used for cancer treatment.

OBJECTIVES: In the 1990s, the discovery of the important role of matrix metalloproteinases (MMPs) in cancer angiogenesis, growth and metastasis galvanised research efforts to search for ways to inhibit these MMPs. To date, this has resulted in the investigation of approximately 50 MMPIs which have undergone various phases of clinical trials. However, despite a large body of research being devoted to discovery and development of MMPIs, results have largely not been supportive of this approach to anticancer treatment. KEY FINDINGS: The reasons for the general failure of these drugs in clinical trials include various unwanted side-effects, the use of healthy volunteers to provide drug dosages which did not correctly reflect dosages for cancer patients, and the exclusion of patients with early stage cancer in clinical trials despite MMPs being determined to be critical for the angiogenic switch, a process associated with early tumour growth. In contrast, a naturally-occurring endogenous protein and a non-functional serine protease inhibitor (serpin), pigment epithelium-derived factor (PEDF), has been proposed for cancer therapy partly due to its ability to regulate specific MMPs central to cancer progression. SUMMARY: PEDF has been found to specifically downregulate membrane-type I matrix metalloproteinase (MT1-MMP) and furthermore, potentially matrix metalloproteinase-2 (MMP-2), two of the most commonly implicated MMPs in neoplasia.

Elevated level of fibrinogen increases caveolae formation; role of matrix metalloproteinase-9.

The role of the inflammatory agent fibrinogen (Fg) in increased pial venular permeability has been shown previously. It was suggested that an activation of matrix metalloproteinase-9 (MMP-9) is involved in Fg-induced enhanced transcytosis through endothelial cells (ECs). However, direct link between Fg, caveolae formation, and MMP-9 activity has never been shown. We hypothesized that at an elevated level, Fg enhances formation of functional caveolae through activation of MMP-9. Male wild-type (WT, C57BL/6J) or MMP-9 gene knockout (MMP9-/-) mice were infused with Fg (4 mg/ml, final blood concentration) or equal volume of phosphate buffered saline (PBS). After 2 h, mice were sacrificed and brains were collected for immunohistochemical analyses. Mouse brain ECs were treated with 4 mg/ml of Fg or PBS in the presence or absence of MMP-9 activity inhibitor, tissue inhibitor of metalloproteinases-4 (TIMP-4, 12 ng/ml). Formation of functional caveolae was assessed by confocal microscopy. Fg-induced increased formation of caveolae, which was defined by an increased co-localization of caveolin-1 (Cav-1) and plasmalemmal vesicle-associated protein-1 and was associated with an increased phosphorylation of Cav-1, was ameliorated in the presence of TIMP-4. These results suggest that at high levels, Fg enhances formation of functional caveolae that may involve Cav-1 signaling and MMP-9 activation.

Novel antiinflammatory therapies for COPD.

The biologic nature of COPD inflammation is not well understood and agents that inhibit inflammation in COPD are a major unmet need. However, a variety of agents that have the potential to be inhibitors of COPD inflammation are in various stages of development. Agents that have been approved for a non-COPD indication but that have potential for inhibiting COPD inflammation include the statins, some phosphodiesterase inhibitors, some long-acting ß agonists, tiotropium bromide, the peroxisome proliferator-activated receptor-γ agonist rosiglitazone, and various monoclonal antibodies. New molecular entities that are being developed specifically as antiinflammatory agents for COPD include a variety of chemokine receptor antagonists, inhibitors of matrix metalloproteinases, inhibitors of p38 mitogen-activated protein kinases, and stem cells. Some other novel agents that are in preclinical or early clinical stages are mentioned.

[Experimental study on effects of matrix metalloproteinase inhibitor on posterior capsule opacification in rabbits].

OBJECTIVE: To determine whether matrix metalloproteinase (MMP) inhibitor can provide therapeutic effects for rabbits posterior capsule opacification in vivo and to observe the side effects of this drug on surrounding intraocular structures. METHODS: Experimental research. New Zealand white rabbits were undertaken phacoemulsification operation. GM6001 at different concentrations (100, 200 and 500 µmol/L) and GM6001 negative control liqueur were infused into the capsule bags of the rabbits at the end of operation and two days after the operation. The incidence of posterior capsule opacification was assessed and the histological sections of posterior capsules were observed under microscope 12 weeks after the surgery. The anterior chamber response was observed on day 2 post-operatively. The changes of intraocular pressure were measured by day 2 and day 7. Corneal endothelial cells were observed under scanning electron microscope and iris, ciliary body and retina were observed under microscope on day 7. RESULTS: GM6001 significantly prevented posterior capsule opacification (P = 0.007). No opacification occurred on the rabbit posterior capsule in eyes with 200 and 500 µmol/L GM6001 on week 12 post-operatively in vivo. No cells were found on posterior capsule in 500 µmol/L group, whereas lens epithelial cells and fibroblasts were found in the controls under microscope. No difference of anterior chamber flare between the eyes with GM6001 at different concentrations and the control group (P = 0.380) by day 2 after the operation. The intraocular pressure in eyes with GM6001 was the same as that in the control 2-days (F = 0.642, P = 0.597) and 7-days (F = 0.179, P = 0.909) post-operation. The corneal endothelial cells in eyes with 500 µmol/L GM6001 arranged regularly and did not show any difference from that in the control eyes under scanning electron microscope 7-day after the operation. The iris, ciliary body and retina in eyes with 500 µmol/L GM6001 were normal in appearance 7-day after the operation. CONCLUSIONS: MMP inhibitor can prevent posterior capsule opacification effectively in rabbits in vivo and does not cause damage to surrounding intraocular structures, suggesting that MMP inhibitor may become a medication used for the prevention of lens posterior capsule opacification.

Effects of formoterol and ipratropium bromide on repeated cadmium inhalation-induced pulmonary inflammation and emphysema in rats.

The anti-inflammatory properties of inhaled formoterol and ipratropium bromide, alone or in combination, were investigated in a rat model of chronic pulmonary inflammation with airspace enlargement induced by cadmium inhalation. At the end of the protocol, cadmium-induced increase of airway resistance was prevented by formoterol (4 mg/30 ml) or ipratropium (0.20 mg/20 ml). Formoterol elicited a significant decrease in total cell and neutrophil counts in bronchoalveolar lavage fluid as well as on the activity of gelatinase B (MMP-9), an enzyme strongly expressed in alveolar macrophages and epithelial cells. Additionally, a significant attenuation of the lung lesions characterized by inflammatory cell infiltration within the alveoli and the interstitium and a decrease in mean linear intercept were observed. Although ipratropium alone had no effects on the cadmium-induced pulmonary inflammation and emphysema, its combination with an inefficient concentration of formoterol (1 mg/30 ml) showed a synergistic inhibitory effect on neutrophil and total cell counts as well as on the mean linear intercept associated with a synergistic inhibition on the MMP-9 activity. Gelatinase A (MMP-2) activity was not influenced by drug pretreatments. Neither macrophage metalloelastase (MMP-12) activity nor levels of cytokines IL-1ß, TNF-α and GM-CSF in bronchoalveolar lavage fluid were modified in rats chronically exposed to cadmium. No desensitization of ß(2)-adrenoceptors or cholinergic receptors on airway smooth muscles and inflammatory cells during the protocol was observed. In conclusion, formoterol alone or combined with ipratropium bromide partially protects the lungs against the chronic inflammation and airspace enlargement by reducing neutrophilic infiltration possibly via the inhibition of MMP-9 activity.

Effect of doxycycline on postoperative scarring after trabeculectomy in an experimental rabbit model.

PURPOSE: To investigate effectiveness of doxycycline after trabeculectomy in rabbits by evaluating bleb appearance, intraocular pressure, and levels of matrix metalloproteinase-1, -2, -3, and -9 and tissue inhibitors of metalloproteinase (TIMP)-1 and -2 in the subconjunctival (sc) area. METHODS: Twenty-nine New Zealand White rabbits were assigned into 1 of 6 groups as follows: topical doxycycline (0.1%), postoperative sc injection of doxycycline (100 mg/2 mL), and intraoperative mitomycin-C (MMC) (0.2 mg/mL) and their respective control groups. RESULTS: There was significant difference between intraocular pressure in the case groups, but there was no significant difference in topical doxycycline and MMC groups during the follow up. In the topical doxycycline group, levels of TIMP-1 and perifericTIMP-1 were higher and levels of perifericMMP-2 and inflammation were lower than their controls. In the sc doxycycline group, peripheral inflammation was higher than in the corresponding control. Only peripheral inflammation was significantly different between case groups, with the highest level in sc and the lowest level in MMC groups. Further, topical doxycycline group showed no significant difference in bleb appearance or peripheral inflammation compared with MMC group. Conjunctival burn and corneal vascularization were detected only in the sc doxycycline group. CONCLUSIONS: Topical doxycycline is more effective than sc doxycycline but is similar to MMC, and it can be an alternative to MMC in trabeculectomy in rabbits.

Stromelysin-1 (MMP-3) is a target and a regulator of Wnt1-induced epithelial-mesenchymal transition (EMT).

Matrix metalloproteinases (MMPs) play a well-defined role in later stages of tumor progression. However, there has been evidence that they also contribute to earlier stages of malignant transformation. The Wnt signaling transduction pathway plays a critical role in development and in the pathogenesis of many epithelial cancers. Here we have used Wnt1-induced epithelial-mesenchymal transition (EMT) in C57MG murine mammary epithelial cells to study the role of MMPs in this early step of malignant progression. Overexpression of Wnt1 in C57MG cells promoted EMT, the translocation of ß-catenin from the cell membrane to the nucleus and its transcriptional activity, cell proliferation and cell motility. Simultaneously, we observed an increased expression of stromelysin-1 (MMP-3) and a 5.5-fold increase in MMP-3 promoter activity in C57MG cells expressing Wnt1 compared with C57MG cells. Treatment of Wnt-overexpressing cells with MMP inhibitor AG3340 decreased MMP-3 expression. We also found evidence that MMP-3 and Wnt3a cooperate in enhancing the transcriptional activity of ß-catenin in C57MG cells. Consistently, the effects of Wnt1 on EMT, proliferation and migration were inhibited by MMP inhibitors, or upon downregulation of MMP-3 by siRNA. These results suggest that MMP-3 is both a direct transcriptional target and a necessary contributor of the Wnt/ß-catenin signaling pathway.