Inhibition of breast cancer cell extracellular matrix degradative activity by chemically modified tetracyclines

BACKGROUND. Inhibition of tumour cell proliferation, invasion and metastasis by chemically modified tetracyclines has been ascribed to inhibition of matrix metalloproteinase (MMP) activity.

METHODS. Exposure of the human breast carcinoma cell line MDA‐MB‐231 or its MMP‐9‐overproducing transfected clone (E‐10) to 6‐demethyl, 6‐deoxy, 4‐de [dimethylamino]‐tetracycline (CMT‐3), a chemically modified non‐antimicrobial tetracycline followed by analysis using gelatinase activity assay, zymography, degradation of radiolabelled extracellular matrix (ECM), Western blotting, TNF‐α ELISA and cell viability assays.

RESULTS. CMT‐3 treatment results in diminution in extracellular MMP‐9 protein levels as well as inhibition of gelatinase activity. This prevents cell‐mediated ECM degradation without inducing general cytostasis or cytotoxicity. Culturing E‐10 cells in 10 or 20?µM CMT‐3 diminished secreted MMP‐9 levels by 45% or 60%, respectively, but did not affect levels of most other secreted proteins, including tissue inhibitor of Metalloproteinases (TIMP‐1). ECM degradation by E‐10 cells or their conditioned medium was inhibited by ～ 20%–30% in the presence of 20?µM CMT‐3, reflecting inhibition of MMP‐9 activity in addition to diminution of released MMP‐9 levels. TNF‐α levels were also diminished in E‐10 conditioned medium in the presence of CMT‐3, but cell viability, measured by MTS reduction and cytosolic LDH retention, was unaffected.

CONCLUSIONS. It is proposed that the reduction in ECM‐degradative activity reflects diminished levels of expression as well as inhibition of enzymatic activity of MMPs released by cells in the presence of CMT‐3. These multiple effects of CMT‐3 may offer promise for use in suppressing tumour invasion, and if used in conjunction with other chemotherapy agents, may lead to more successful treatment of cancer.     

Inhibition of matrix metalloproteinases by chemically modified tetracyclines in sepsis

Sepsis precipitates a systemic inflammatory stimulus that causes systemic release of cytokines and sequestration of polymorphonuclear neutrophils, resulting in degranulation of matrix metalloproteinases (MMPs), which causes extracellular matrix basement membrane degradation. One of the important anti-inflammatory properties of tetracyclines is their ability to inhibit MMPs. In this study, we focused on the regulation of MMPs in sepsis and their reduction by treatment with nonantimicrobial chemically modified tetracyclines (CMTs), which retain their anti-inflammatory activity. Sepsis was induced by cecal ligation and puncture (CLP) method. At 24 h and 1 h before CLP, some rats received CMT-3 (25 mg/kg), another group of rats received hydroxamate (H; an inhibitor of MMP; 25 mg/kg), and untreated rats received saline by gavage. At 0 h, 0.5 h, 1.5 h, and 24 h after CLP, blood and liver samples were collected. Plasma and liver MMP-9 by zymography and Western immunoblotting, plasma nitric oxide by measuring nitrate level, plasma glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) by enzymatic method, and liver gelatinase by radiolabeled gelatin lysis assay and 24 h mortality were determined. Plasma MMP-9 (92 kDa), nitrate, and GOT and GPT levels were elevated compared with the time 0 level and reached peak at 1.5 h CLP and remained high for 24 h. Both CMT-3 and H treatment reduced GOT,GPT, 92-kDa gelatinase, and nitrate levels throughout the 24 h. CMT-3 and H are equally effective in sepsis treatment. The 24-h mortality for CLP rats was 30%, whereas pretreatment with CMT-3 and H resulted in 0% mortality. Hepatic MMP-9 and gelatinase activity increased significantly after CLP, and pretreatment with CMT-3 and H inhibited these expressions. These results indicate the beneficial effect of CMT-3 in preventing the increase in GOT, GPT, NO, MMP-9, gelatinase activity, and the ensuing septic shock.     

Effect of chemically modified tetracycline on transforming growth factor-beta1 and caspase-3 activation in liver of septic rats

OBJECTIVES: We have previously demonstrated that hepatic matrixmetalloproteinase (MMP)-9 and gelatinase activity increased significantly after sepsis, and pretreatment with chemically modified tetracycline (CMT-3) inhibited these expressions and improved survivability. It has been established that MMP-9 release from hepatic nonparenchymal cells activates transforming growth factor (TGF)-beta1, which in turn catalyzes the conversion of procaspase-8 into active caspase-8. Caspase-8 activates caspase-3, which in turn degrades fibronectin and focal adhesion kinase and leads to disruption of hepatic architecture and integrity. We have been interested in investigating the role of posttreatment with CMT-3 on hepatic MMP-9, TGF-beta1, and caspase-3 activity following sepsis. DESIGN: Laboratory experiment. SETTING: University laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: In this study, sepsis was induced in rats by cecal ligation and puncture (CLP), and 2 hrs later, half of the rats received CMT-3 (25 mg/kg), whereas the other half received vehicle by gavage. Twenty-four and 48 hrs after sepsis induction, blood and liver samples were collected. MEASUREMENTS AND MAIN RESULTS: Plasma glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) levels were determined by enzymatic method, and the activation states of hepatic MMP-9, MMP-2, tissue inhibitor of metalloproteinase (TIMP)-1, TGF-beta1, and caspase-3 were determined by Western immunoblotting. Plasma GOT, GPT, and hepatic MMP-9 activity increased 2.5-fold, and TFG-beta1 and caspase-3 activity increased 1.5- to 2-fold at 24 hrs and 48 hrs post-CLP; CMT-3 treatment blocked these increases. Furthermore, CMT-3 treatment also led to increased TIMP-1 level, an in vivo inhibitor of MMP-9. MMP-2 level was unaffected by CLP. The 24-hr and 48-hr mortality rates for CLP rats were 29% and 50%, whereas posttreatment with CMT-3 resulted in 0% mortality. CONCLUSIONS: Our results are consistent with an MMP-9-induced caspase-3 activation in response to CLP. CMT-3 posttreatment increased TIMP-1 level and thereby inhibited MMP-9, which in turn decreased TGF-beta1 and caspase-3 signaling pathways and improved survivability in septic rats.     

Lacidipine [correction of Lalsoacidipine] modulates the secretion of matrix metalloproteinase-9 by human macrophages

Activated macrophages within the arterial wall secrete matrix-degrading metalloproteinases (MMPs) that weaken the atherosclerotic plaque and contribute to its fissuration. Preclinical studies have shown that calcium antagonists may reduce atherogenesis in the arterial wall. In the present study we evaluated the effect of lacidipine on 92-kDa gelatinase B (MMP-9) expression in human macrophages in cultures. Cells were treated for 24 h with lacidipine and the conditioned media were analyzed. Lacidipine (1-20 microM) significantly reduced, in a dose-dependent manner, MMP-9 potential gelatinolytic capacity up to 50%. When MMP-9 expression was stimulated by treatment with phorbol esters or tumor necrosis factor-alpha, lacidipine was able to inhibit this enhanced gelatinolytic capacity up to 50 and 60%, respectively. Western blot analysis and enzyme-linked immunosorbent assay showed a reduction of MMP-9 protein actually released by cells. The addition of lacidipine in the incubation media determined no significant variation in Ca(2+) concentration. The drug did not affect MMP-9 mRNA levels, but it effectively reduced the amount of both active and total free MMP-9 secreted by human macrophages. Lacidipine reduced also the secretion of the tissue inhibitor of metalloproteinase-1 (TIMP-1); however we observed an overall reduction of the gelatinolytic activity of the cells. Finally, peritoneal macrophages, obtained from mice treated with lacidipine, showed a reduced secretion of MMP-9. Together, our data indicate that lacidipine may potentially exert an antiatherosclerotic activity by modulating the secretion of MMP-9 by macrophages. This, in addition to the previously demonstrated inhibition of cholesterol esterification, may contribute to increase plaque stability.     

Cyclooxygenase inhibition limits blood-brain barrier disruption following intracerebral injection of tumor necrosis factor-alpha in the rat

Increased permeability of the blood-brain barrier (BBB) is important in neurological disorders. Neuroinflammation is associated with increased BBB breakdown and brain injury. Tumor necrosis factor (TNF)-alpha is involved in BBB injury and edema formation through a mechanism involving matrix metalloproteinase (MMP) up-regulation. There is emerging evidence indicating that cyclooxygenase (COX) inhibition limits BBB disruption following ischemic stroke and bacterial meningitis, but the mechanisms involved are not known. We used intracerebral injection of TNF-alpha to study the effect of COX inhibition on TNF-alpha-induced BBB breakdown, MMP expression/activity, and oxidative stress. BBB disruption was evaluated by the uptake of (14)C-sucrose into the brain and by magnetic resonance imaging utilizing gadolinium-diethylenetriaminepentaacetic acid as a paramagnetic contrast agent. Using selective inhibitors of each COX isoform, we found that COX-1 activity is more important than COX-2 in BBB opening. TNF-alpha induced a significant up-regulation of gelatinase B (MMP-9), stromelysin-1 (MMP-3), and COX-2. In addition, TNF-alpha significantly depleted glutathione as compared with saline. Indomethacin (10 mg/kg i.p.), an inhibitor of COX-1 and COX-2, reduced BBB damage at 24 h. Indomethacin significantly attenuated MMP-9 and MMP-3 expression and activation and prevented the loss of endogenous radical scavenging capacity following intracerebral injection of TNF-alpha. Our results show for the first time that BBB disruption during neuroinflammation can be significantly reduced by administration of COX inhibitors. Modulation of COX in brain injury by COX inhibitors or agents modulating prostaglandin E(2) formation/signaling may be useful in clinical settings associated with BBB disruption.     

Gelatinase activities in wounds of healing-impaired mice versus wounds of non-healing-impaired mice

The gelatinases, matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9), have been implicated in different aspects of wound repair. However, little is known about MMP-2 and MMP-9 activity in animal models of impaired wound healing. We sought to compare serial gelatinase activities for 25 days after full-thickness excisional wounds in genetically diabetic healing-impaired mice and their nondiabetic non-healing-impaired littermates. Wound samples were frozen, homogenized, clarified by centrifugation, and analyzed on zymography gels, and MMP bands were quantitated relative to a conditioned media standard from HT-1080 cells. Gelatinase activity in both diabetic mice and nondiabetic mice increased after the mice were wounded. However, levels of latent gelatinases peaked earlier in the diabetic wounds, and there was more active MMP-2 and MMP-9 in the wounds of the diabetic mice than in the wounds of the nondiabetic mice. Because the higher gelatinase activity in the wounds of the diabetic mice was similar to the higher levels of gelatinase reported in difficult-to-heal wounds such as ulcers and burns, this diabetic mouse model may be useful for studies of these proteinases and their inhibitors in impaired wound healing.     

A chemically modified tetracycline (CMT-3) is a new antifungal agent

Several chemically modified tetracycline analogs (CMTs), which were chemically modified to eliminate their antibacterial efficacy, were unexpectedly found to have antifungal properties. Of 10 CMTs screened in vitro, all exhibited antifungal activities, although their efficacies varied. Among these compounds, CMT-315, -3, and -308 were found to be the most potent as antifungal agents. The MICs of CMT-3 against 47 strains of fungi in vitro were determined by using amphotericin B (AMB) and doxycycline as positive and negative controls, respectively. The MICs of CMT-3 were generally found to be between 0.25 and 8.00 microg/ml, a range that approximates the blood levels of this drug when administrated orally to humans. Of all the yeast species tested to date, Candida albicans showed the greatest sensitivity to CMT-3. The filamentous species most susceptible to CMT-3 were found to be Epidermophyton floccosum, Microsporum gypseum, Pseudallescheria boydii, a Penicillium sp., Scedosporium apiospermum, a Tricothecium sp., and Trichophyton rubrum. Growth inhibition of C. albicans by CMT-3, determined by a turbidity assay, indicated a 50% inhibitory concentration of 1 microg/ml. Thirty-nine strains, including 20 yeasts and 19 molds, were used to measure viability (the ability to grow after treatment with a drug) inhibition by CMT-3 and AMB. CMT-3 exhibited fungicidal activity against most of these fungi, especially the filamentous fungi. Eighty-four percent (16 of 19) of the filamentous fungi tested showed more than 90% inhibition of viability by CMT-3. In contrast, AMB showed fungicidal activity against all yeasts tested. However, most of the filamentous fungi (16 of 19) showed less than 50% inhibition of viability by AMB, indicating that AMB is fungistatic against most of these filamentous fungi. To begin to identify the sites in fungal cells affected by CMT-3, C. albicans and a Penicillium sp. were incubated with the compound at 35 degrees C, and then the fluorescence of CMT-3 was observed by confocal laser scanning electron microscopy. CMT-3 appeared to have widespread intracellular distribution throughout C. albicans and the Penicillium sp. The mechanisms of the antifungal activity of CMT-3 are now being explored.     

Basement Membrane and Repair of Injury to Peripheral Nerve: Defining a Potential Role for Macrophages, Matrix Metalloproteinases, and Tissue Inhibitor of Metalloproteinases-1

Injury to a peripheral nerve is followed by a remodeling process consisting of axonal degeneration and regeneration. It is not known how Schwann cell–derived basement membrane is preserved after injury or what role matrix metalloproteinases (MMPs) and their inhibitors play in axonal degeneration and regeneration. We showed that the MMPs gelatinase B (MMP-9), stromelysin-1 (MMP-3), and the tissue inhibitor of MMPs (TIMP)-1 were induced in crush and distal segments of mouse sciatic nerve after injury. TIMP-1 inhibitor activity was present in excess of proteinase activity in extracts of injured nerve. TIMP-1 protected basement membrane type IV collagen from degradation by exogenous gelatinase B in cryostat sections of nerve in vitro. In vivo, during the early phase (1 d after crush) and later phase (4 d after crush) after injury, induction of TNF-α and TGF-β1 mRNAs, known modulators of TIMP-1 expression, were paralleled by an upregulation of TIMP-1 and gelatinase B mRNAs. At 4 days after injury, TIMP-1, gelatinase B, and TNF-α mRNAs were localized to infiltrating macrophages and Schwann cells in the regions of nerve infiltrated by elicited macrophages. TIMP-1 and cytokine mRNA expression was upregulated in undamaged nerve explants incubated with medium conditioned by macrophages or containing the cytokines TGF-β1, TNF-α, and IL-1α. These results show that TIMP-1 may protect basement membrane from uncontrolled degradation after injury and that cytokines produced by macrophages may participate in the regulation of TIMP-1 levels during nerve repair.     

Reactive oxygen species produced by macrophage-derived foam cells regulate the activity of vascular matrix metalloproteinases in vitro. Implications for atherosclerotic plaque stability.

Vulnerable areas of atherosclerotic plaques often contain lipid-laden macrophages and display matrix metalloproteinase activity. We hypothesized that reactive oxygen species released by macrophage-derived foam cells could trigger activation of latent proforms of metalloproteinases in the vascular interstitium. We showed that in vivo generated macrophage foam cells produce superoxide, nitric oxide, and hydrogen peroxide after isolation from hypercholesterolemic rabbits. Effects of these reactive oxygens and that of peroxynitrite, likely to result from simultaneous production of nitric oxide and superoxide, were tested in vitro using metalloproteinases secreted by cultured human vascular smooth muscle cells. Enzymes in culture media or affinity-purified (pro-MMP-2 and MMP-9) were examined by SDS-PAGE zymography, Western blotting, and enzymatic assays. Under the conditions used, incubation with xanthine/xanthine oxidase increased the amount of active gelatinases, while nitric oxide donors had no noticeable effect. Incubation with peroxynitrite resulted in nitration of MMP-2 and endowed it with collagenolytic activity. Hydrogen peroxide treatment showed a catalase-reversible biphasic effect (gelatinase activation at concentrations of 4 microM, inhibition at > or = 10-50 microM). Thus, reactive oxygen species can modulate matrix degradation in areas of high oxidant stress and could therefore contribute to instability of atherosclerotic plaques.     

Role of chemically modified tetracycline on TNF-alpha and mitogen-activated protein kinases in sepsis

Chemically modified tetracyclines are orally active inhibitors of multiple proteases and cytokines. In this study, we focused on the regulation of tumor necrosis factor (TNF)-alpha and mitogen-activated protein kinases (MAPKs) in sepsis and their reduction by treatment with nonantimicrobial chemically modified tetracycline-3 (CMT-3), which retains their antiinflammatory activity. Sepsis was induced in rats by cecal ligation and puncture (CLP). At 24 h and 1 h before CLP, treated rats received CMT-3 (25 mg/kg), and untreated rats received saline by gavage. At 0 h, 0.5 h, 1.5 h, and 24 h after CLP, blood and liver samples were collected. TNF-alpha was determined by ELISA, and MAPKs were determined by Western blot analysis. A significant activation of p38 MAPK was observed after 0.5 h and 1.5 h of sepsis that appeared to coincide with the increased circulating TNF-alpha level. The activation of p42/44 was increased after 24 h of sepsis, whereas that of SAPK/JNK was unaltered throughout the course of sepsis. CMT-3 pretreatment inhibited the TNF-alpha level as well as p38 MAPK activation seen after 0.5 and 1.5 h of CLP and also suppressed the activation of p42/44 after 24 h post-CLP. These results indicate increased activity of TNF-alpha and MAPK following sepsis and demonstrate the beneficial effect of CMT-3 in preventing the increase in TNF-alpha, p38 MAPK, p42/44 MAPK, and the progression of septic shock.     

Peptidoglycan of S. aureus causes increased levels of matrix metalloproteinases in the rat

Matrix metalloproteinases (MMPs) have been suggested to contribute to the organ injury in septic patients. We recently demonstrated that peptidoglycan (PepG) of S. aureus causes organ injury in the rat. A possible role for MMPs in the septic response to PepG is unknown. In the present study, we have examined whether the release of MMP-9 (gelatinase B) and MMP-2 (gelatinase A) is induced by PepG in the anesthetized rat. Male Wistar rats were injected intravenously with PepG (10 mg/kg), LPS (1 mg/kg), or a combination of LPS and PepG (1 mg/kg of each). After 1 or 3 h, liver, lung, and plasma were harvested. MMP-9 and MMP-2 levels were analyzed in organ homogenates and in plasma samples by zymography. MMP-9 levels were significantly increased in the lung within 1 h after injection of PepG, LPS, or combined treatment, compared with sham animals (P < or = 0.05). In the liver and plasma, MMP-9 was clearly increased by PepG or LPS at both 1 and 3 h compared with sham animals (P < or = 0.05). Considerable basal amounts of MMP-2 protein were seen in the liver and in plasma. In the lung, MMP-2 levels were elevated by combined LPS/PepG at 1 h and by LPS at 3 h (P < or = 0.05). In contrast, MMP-2 activity in the liver was significantly reduced by bacterial products. In the plasma, no major alterations of MMP-2 levels were observed. Our data show that PepG of S. aureus causes a rapid elevation of MMP-9 protein in the liver, lung, and blood of the rat. Based on these and previous data, we hypothesize that the release of MMP-9 in lung, liver, and blood is part of the septic host response to systemic PepG.     

明胶酶A、B在脐血CD34+细胞及部分白血病细胞系中的表达

目的　研究骨髓及脐血CD34+ 细胞及白血病细胞系的明胶酶 ,即基质金属蛋白酶 9(MMP 9,明胶酶B)和基质金属蛋白酶 2 (MMP 2 ,明胶酶A)的产生 ,探讨它们在CD34+ 细胞迁移、归巢中的意义及在白血病发病中的作用。方法　通过MiniMACS磁珠分离技术分离脐血及骨髓的CD34+细胞 ,通过酶谱分析法测定脐血、骨髓CD34 + 细胞及白血病细胞系的无血清条件培养液中明胶酶的表达。结果　在脐血CD34+ 细胞的条件培养液中可测出相对分子质量为 92× 10 3的亮带 ,骨髓CD34+ 细胞的条件培养液中未测到亮带。部分白血病细胞系U937、KG 1a、HL 6 0可产生相对分子质量为 92×10 3和 72× 10 3的亮带 ,J6 1、J6 2细胞系可产生相对分子质量为 92× 10 3的亮带 ,而HEL、Namalva、CEM、K5 6 2、LCL H不产生亮带。结论　脐血CD34+ 细胞可产生MMP 9,而骨髓CD34+ 细胞不产生 ,脐血CD34+ 细胞具有较强的穿透基底膜的迁移能力并与MMP 9相关。在白血病细胞系中 ,部分髓系白血病细胞系产生MMP 9和MMP 2 ,而非髓系白血病细胞系不产生。     

Matrix metalloproteinases: potential therapeutic target in spinal cord injury.

Mediators of extracellular matrix proteins degradation, the matrix metalloproteinases (MMPs), involved in inflammation as well as facilitation of process outgrowth of oligodendrocytes are interesting targets for neural repair. Recent data reported their activation after seizures, cerebral ischemia and spinal cord injury. The present study was designed to localize at cellular level the gelatinase activity by in situ zymography in a rat spinal cord contusion model. The kinetic of gelatinase activation was monitored by in situ zymography on 20 microm cryostat sections. The fluorescein-quenched DQ gelatin digestion yielded cleaved fluorescent peptides enabling the detection of gelatinase activity at cellular level. Twenty four hours and 48 h after injury, a strong gelatinase activity was detected at the lesion site in and around vascular structures and infiltrated cells. A preincubation with either MMP-2 or MMP-9 antibodies significantly decreases the gelatinase activity pattern, suggesting the involvement of at least both MMPs. Our results are consistent with a role for MMPs in the blood spinal barrier disruption, the leukocytes infiltration, the disruption of the extracellular matrix and the clearance of debris.     

Production and localization of 92-kilodalton gelatinase in abdominal aortic aneurysms. An elastolytic metalloproteinase expressed by aneurysm-infiltrating macrophages.

Abdominal aortic aneurysms (AAA) are characterized by disruption and degradation of the elastic media, yet the elastolytic proteinases involved and their cellular sources are undefined. We examined if 92-kD gelatinase, an elastolytic matrix metalloproteinase, participates in the pathobiology of AAA. Gelatin zymography of conditioned medium from normal, atheroocclusive disease (AOD), or AAA tissues in organ culture showed that all tissues produced 72-kD gelatinase. AOD and AAA cultures also secreted 92-kD gelatinase, but significantly more enzyme was released from AAA tissues. ELISA confirmed that AAA tissues released approximately 2-fold more 92-kD gelatinase than AOD tissue and approximately 10-fold more than normal aorta. Phorbol ester induced a 5.3-fold increase in 92-kD gelatinase secretion by normal aorta and AOD and an 11.5-fold increase by AAA. By immunohistochemistry, 92-kD gelatinase was not detected in normal aorta and was only occasionally seen within the neointimal lesions of AOD tissue. In all AAA specimens, however, 92-kD gelatinase was readily localized to numerous macrophages in the media and at the adventitial-medial junction. The expression of 92-kD gelatinase mRNA by aneurysm-infiltrating macrophages was confirmed by in situ hybridization. These results demonstrate that diseased aortic tissues secrete greater amounts of gelatinolytic activity than normal aorta primarily due to increased production of 92-kD gelatinase. In addition, the localization of 92-kD gelatinase to macrophages in the damaged wall of aneurysmal aortas suggests that chronic release of this elastolytic metalloproteinase contributes to extracellular matrix degradation in AAA.     

Secretory leukocyte protease inhibitor suppresses the production of monocyte prostaglandin H synthase-2, prostaglandin E2, and matrix metalloproteinases.

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor found in fluids lining mucosal surfaces. In addition to its primary function as an antiprotease, SLPI may also influence cellular functions associated with enzyme synthesis and retroviral infection. In this study, SLPI was examined for its effect on signaling events involved in the production of matrix metalloproteinases (MMPs) by monocytes. Addition of SLPI before stimulation with concanavalin A or LPS resulted in a significant inhibition of monocyte prostaglandin H synthase-2 (PGHS-2), a pivotal enzyme in the PGE2-cAMP dependent pathway of monocyte MMP synthesis. Suppression of PGHS-2 was detected with 0.1 microg/ml of SLPI with a substantial inhibition at 1 and 10 micro/ml. Attenuation of PGHS-2 by SLPI was accompanied by decreased production of PGE2 resulting in the suppression of interstitial collagenase (MMP-1) and gelatinase B (MMP-9) that was reversed by PGE2 or Bt2cAMP. The inhibitory effect of SLPI was largely independent of its antiprotease activity because SLPI muteins, with significantly lower antiprotease activity, also suppressed the induction of PGHS-2 and MMPs. The inhibitory effects of SLPI did not involve the modulation of monokine production since TNF-alpha and IL-10 were unaffected. These findings demonstrate that SLPI also functions as a potent antiinflammatory agent by interfering with the signal transduction pathway leading to monocyte MMP production.     

The selective phosphodiesterase 4 inhibitor RP 73-401 reduced matrix metalloproteinase 9 activity and transforming growth factor-beta release during acute lung injury in mice: the role of the balance between Tumor necrosis factor-alpha and interleukin-10

Matrix metalloproteinases (MMPs) and transforming growth factor (TGF)-beta are involved in airway remodeling associated with the inflammatory process. In this study, we investigated the effect of RP 73-401 (piclamilast), a selective phosphodiesterase-4 inhibitor, on MMP-9 activity and TGF-beta production in two murine models of acute inflammation. In the first model, the lipopolysaccharide (LPS)-induced increase in neutrophils, MMP-9 activity, and tumor necrosis factor (TNF)-alpha and TGF-beta release in bronchoalveolar lavage (BAL) was significantly reduced by RP 73-401 pretreatment. In contrast, the BAL interleukin (IL)-10 level was decreased by LPS but restored by RP 73-401. IL-10 administration in LPS-exposed mice elicited a significant reduction in BAL neutrophilia, MMP-9 activity, and TNF-alpha release but not in TGF-beta production. In the second model, RP 73-401 inhibited BAL neutrophils but not MMP-9 activity and TGF-beta production that were induced by intranasal TNF-alpha. We demonstrated that RP 73-401 might modulate the expression of airway remodeling-associated mediators such as MMP-9 and TGF-beta and that this effect seemed to be at least partially mediated by the balance between TNF-alpha and IL-10.     

Effect of angiotensin II on primary cardiac fibroblast matrix metalloproteinase activities

Left ventricular dysfunction is associated with reperfusion injury occurring during open-heart surgery. There is an increased secretion of angiotensin II (Ang II) and increased matrix metalloproteinases (MMPs) activities associated with open-heart surgery that may affect the cardiac extracellular matrix (ECM). The goal of this study was to determine the effects of Ang II and selective angiotensin II receptor (AT1-R and AT2-R) blockers on the enzymatic activities of MMPs in primary adult murine cardiac fibroblasts (CF). Our hypothesis is that Aug II, with and without a selective receptor blocker, differentially affects CF MMPs activities. The CF were treated with Ang II (10(-6) M) and doses of AT1-R and AT2-R blockers (losartan and PD123319, respectively) at doses of 10(-7) to 10(-5) M for 48 hours. The Ang II-stimulated CF reduced collagenase activities by only 24% (p = 0.004); however, the MMP-2 and MMP-9 gelatinase activities were reduced by 42% and 39%, respectively (p = 0.022). The losartan dose dependently increased MMP-2 (p = 0.02) and MMP-9 (ns). PD123319 at 10(-5) M significantly reduced MMP-2 and MMP-9 activities compared with the Ang II group (p = 0.014 and p = 0.02, respectively). The doses of PD123319 at 10(-6) and 10(-7) M increased the MMP-2 and MMP-9 enzymatic activities significantly above the Ang II only group. Thus, Ang II and AT1-R and AT2-R differentially affect the collagenase and gelatinase MMPs activities released by cardiac fibroblasts.     

Tissue factor pathway inhibitor-2 is a novel inhibitor of matrix metalloproteinases with implications for atherosclerosis

Degradation of ECM, particularly interstitial collagen, promotes plaque instability, rendering atheroma prone to rupture. Previous studies implicated matrix metalloproteinases (MMPs) in these processes, suggesting that dysregulated MMP activity, probably due to imbalance with endogenous inhibitors, promotes complications of atherosclerosis. We report here that the serine proteinase inhibitor tissue factor pathway inhibitor-2 (TFPI-2) can function as an MMP inhibitor. TFPI-2 diminished the ability of the interstitial collagenases MMP-1 and MMP-13 to degrade triple-helical collagen, the primary load-bearing molecule of the ECM within human atheroma. In addition, TFPI-2 also reduced the activity of the gelatinases MMP-2 and MMP-9. In contrast to the "classical" tissue inhibitors of MMPs (TIMPs), TFPI-2 expression in situ correlated inversely with MMP levels in human atheroma. TFPI-2 colocalized primarily with smooth muscle cells in the normal media as well as the plaque's fibrous cap. Conversely, the macrophage-enriched shoulder region, the prototypical site of matrix degradation and plaque rupture, stained only weakly for TFPI-2 but intensely for gelatinases and interstitial collagenases. Evidently, human mononuclear phagocytes, an abundant source of MMPs within human atheroma, lost their ability to express this inhibitor during differentiation in vitro. These findings establish a new, anti-inflammatory function of TFPI-2 of potential pathophysiological significance for human diseases, including atherosclerosis.