Total Matrix Metalloproteinase Research Articles

Effect of okam on inflammation and remodeling of airway of mice with ovalbumin induced asthma

To investigate the effect of okam on inflammation and remodeling of airway in mice with ovalbumin (OVA) induced asthma. Thirty-two mice of Kunming strain were divided into four groups randomly: model group, glucocorticoid inhalation group, okam group and control group, with 8 mice in each group. The asthmatic mice model was reproduced by combined injection and aerosol inhalation of OVA. The mice in model group received normal saline (0.3 ml) gavage daily. The mice in glucocorticoid inhalation group received budesonide (0.4 ml, 200 mug) and normal saline (3.6 ml) inhalation. The mice in okam group were gavaged with okam daily (50 mg/kg). The controls were given normal saline instead of OVA sensitization. All mice were sacrificed 42 days later, followed by lavage of tracheo-bronchial tree of the right lung, and the right lung was saved for pathological examination. The total cell number and differentiation in bronchoalveolar lavage fluid (BALF) were counted under microscope. The expression of interferon-gamma (IFN-gamma), interleukin-4 (IL-4) in BALF were assessed by enzyme linked immunosorbent assay (ELISA). The histological changes in the bronchi and alveoli were evaluated after hematoxylin and eosin (HE) staining. The expression of matrix metalloproteinase-9 (MMP-9) as well as the tissue inhibitor of metalloproteinase-1 (TIMP-1) were determined by immunohistochemistry. Compared with the model group, the total cell count and IL-4 level in BALF, and the score of pathological changes in the broncho-alveolar tissue in okam group or glucocorticoid inhalation group were lower significantly, and the IFN-gamma level elevated markedly (all P<0.01). The MMP-9, TIMP-1 expression in glucocorticoid inhalation group and the TIMP-1 expression in okam group were decreased greatly (P<0.05 or P<0.01). All of above indexes showed marked differences between control group and okam group (P<0.05 or P<0.01). There were significant changes in the total cell count, IFN-gamma, pathological changes, MMP-9 and TIMP-1 between the glucocorticoid inhalation group and the okam group (P<0.05 or P<0.01). Okam may alleviate inflammation of the bronchial and degrade the development of airway remodeling to some degree.

Influence of cytokine inhibitors on concentration and activity of MMP-1 and MMP-3 in disc herniation

IntroductionSpontaneous resorption of disc herniation (DH) after sciatica is well documented. The matrix metalloproteinases (MMP)-1 and MMP-3 are enzymes potentially involved in this process. Glucocorticoid injections are commonly used for treatment, and other anti-inflammatory molecules like tumor necrosis factor (TNF) inhibitors are under clinical investigation. However, little is known about the effect of these molecules on DH resorption.MethodsDH tissue was harvested from patients undergoing surgery for sciatica. Samples were thoroughly washed. Diced explants were cultured ex-vivo in 1) 0.5 ml Dulbecco's modified Eagle's medium (DMEM) 10% fetal calf serum (FCS), (controls), 2) recombinant interleukin 1 receptor antagonist (IL-1Ra), (100 ng/ml), 3) dexamethasone (10E-5 M), or 4) TNF inhibitor monoclonal antibody (10 μg/ml). Supernatants were harvested at 48 hours and frozen. Immunocapture activity assays determined total MMP activity, active MMP levels and pro-MMP levels.ResultsFourteen DH tissue samples were analysed. Levels of all forms of MMP-3 were higher than the respective levels of MMP-1(P < 0.01). In particular, the median (interquartile range [IQR]) total MMP-3 level was 0.97 (0.47 - 2.19) ng/mg of tissue compared to 0.024 (0.01 - 0.07) ng/mg of total MMP-1 level (P < 0.01). Incubation with IL-1Ra, dexamethasone, or TNF inhibitors significantly decreased levels of all forms of MMP-3 (P < 0.05). Dexamethasone significantly decreased the ratio of active MMP-3 to total MMP-3 activity. A significant inhibitory effect of dexamethasone was observed only on active MMP-1, while IL-1 and TNF inhibitor had no significant effect on any form.ConclusionsMMP-3 appears to play a greater role than MMP-1 in DH resorption. Dexamethasone, IL-1-Ra and TNF inhibitor decreased active MMP-3, indicating that the clinical use of these drugs may affect the resorption of DH under certain conditions.

Inhibition of Matrix Metalloproteinases Enhances In Vitro Repair of the Meniscus

Damage or injury of the meniscus is associated with onset and progression of knee osteoarthritis (OA). The intrinsic repair capacity of the meniscus is inhibited by inflammatory cytokines, such as interleukin-1 (IL-1). Using an in vitro meniscal repair model system, we examined the hypothesis that inhibition of matrix metalloproteinases (MMPs) in the presence of IL-1 will enhance repair of meniscal lesions. Integrative repair of the meniscus was examined between two concentric explants cultured with IL-1 and various MMP inhibitors for 14 days. Throughout the culture period, we assessed total specific MMP activity in the media. At harvest, biomechanical testing to assess the strength of repair and histologic staining were performed. IL-1 decreased the shear strength of repair, as compared with control explants. In the presence of IL-1, the broad-spectrum MMP inhibitor GM 6001 decreased the MMP activity in the media, increased the shear strength of repair, and enhanced tissue repair in the interface. However, individual MMP inhibitors did not alter the shear strength of repair in either the presence or absence of IL-1. These findings suggest IL-1 may inhibit meniscal repair through upregulation of MMPs, but inhibition of multiple MMPs may be necessary to promote integrative meniscal repair.

Collagenolytic Fragments and Active Gelatinase Complexes in Periodontitis

Periodontal tissues remodel rapidly, which enables quick adaptation to mechanical changes. Matrix metalloproteinases (MMPs) are involved in these remodeling processes under control of tissue inhibitor of metalloproteinases (TIMPs). In periodontitis, overactivity of MMPs results in pathologic tissue degradation. The aim of this study was to analyze MMPs and TIMPs in healthy and diseased gingiva, periodontal ligament (PDL), and gingival crevicular fluid (GCF). Samples of gingiva, PDL, and GCF were obtained from healthy controls (gingiva: n = 18; PDL: n = 15; GCF: n = 8) and subjects with periodontitis (gingiva: n = 11; PDL: n = 18; GCF: n = 12). MMPs and TIMPs were analyzed by gelatin-, collagen-, and reverse zymography and by Western blotting. Total MMP activity was analyzed using a fluorogenic substrate. TIMP-1 and -2, active and pro-MMP-2 and -9, and active MMP-1 and -8 were present in all samples. Large amounts of active MMP-2 complexes and collagenolytic fragments were also found. Their levels were higher in PDL and GCF from subjects with periodontitis. In general, TIMP levels were lower in diseased periodontal tissues. Especially diseased GCF contained more MMPs. Surprisingly, some MMPs were more abundant in healthy gingiva and PDL than in diseased tissue. Unexpected variations in MMP and TIMP levels in gingiva, PDL, and GCF may result from differences in subject characteristics and disease activity. The levels of active MMP-2 complexes and collagenolytic fragments are higher in the periodontium of subjects with periodontitis and might contribute significantly to periodontal destruction.

Pravastatin increases the expression of the tissue inhibitor of matrix metalloproteinase-1 and the oncogeneBaxin human aortic abdominal aneurysms

The effect of pravastatin on matrix metalloproteinase-9 (MMP-9) and the level of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 was studied in explants of human abdominal aortic aneurysm (AAA) obtained from 13 patients. The effect of pravastatin on the apoptotic status of human AAA explants was also examined. Total MMP-9 content did not differ in human AAA explants incubated in vitro in the presence or absence of pravastatin (10-6 mol/L) for 48 h. TIMP-1 levels were significantly increased in pravastatin-incubated AAA explants, but TIMP-2 production was not modified by pravastatin. Western blot experiments showed that, whereas Bax expression was increased in pravastatin-incubated AAA explants, the expression of Bcl-2 was not modified. On the other hand, the ratio of the expression of Bax to Bcl-2, an apoptotic index, was not modified by pravastatin. In the human AAA explants, the increase in Bax expression, but not the increase in TIMP-1 expression elicited by pravastatin, was reversed by L-mevalonate, a downstream HMG-CoA reductase metabolite, suggesting that the expression of Bax and TIMP-1 followed HMG-CoA reductase-dependent and -independent pathways, respectively. In conclusion, pravastatin increases both TIMP-1 and Bax expression in human AAA explants without changes in either MMP-9 activity or the apoptotic status.

Matrix metalloproteinase–9 promoter polymorphisms in Korean patients with systemic lupus erythematosus

To investigate the association between functional promoter polymorphisms of matrix metalloproteinase-9 (MMP-9) and systemic lupus erythematosus (SLE), we analyzed MMP-9 promoter -1562 C>T and MMP-9 -90 (CA)(n) repeat polymorphisms in 135 Korean SLE patients (mean age, 34.7 years; 124 female and 11 male) and in 135 gender- and age-matched healthy controls (mean age, 35.4 years). Clinical and laboratory findings were collected during the follow-up period (mean, 63.5 months; range, 3-252 months), and Systemic Lupus International Collaborating Clinics/American College of Rheumatology (SLICC/ACR) Damage Indexes were calculated. The levels of total MMP-9 were measured in sera of SLE patients and controls by enzyme-linked immunoabsorbent assay. The serum levels of MMP-9 in SLE patients were significantly lower than those of controls (mean +/- standard error of the mean, 1421.6+/-177.4 vs 3731.4+/-441.4 ng/ml, p=1.2 x 10(-5) by t test). Both functional polymorphisms were under the Hardy-Weinberg equilibrium state except (CA)(n) repeat polymorphisms in SLE patients (p=2.6 x 10(-5) by chi(2) goodness-of-fit test). The distribution of the MMP-9 promoter polymorphisms or haplotypes was not significantly different in SLE patients and controls. However the frequency of alleles with low numbers of CA repeats (n<21, 11.9% vs 7.0%, p=0.06 by the chi(2) test; odds ratio=1.78, 95% confidence interval=0.99-3.20) and the prevalence of low CA repeats homozygote tended to be higher in patients than in controls (5.2% vs 0.7%, p=0.07 by logistic regression, odds ratio=7.29, 95% confidence interval=0.88-60.10) in the recessive model. No relationship was found between MMP-9 polymorphisms and clinical features or damage as indicated by SLICC/ACR Damage Index in the study subjects. These results suggest that genetic polymorphisms of the MMP-9 promoter regions are not associated with the development of SLE in Korea.

Circulating matrix metalloproteinase 9 levels in relation to sampling methods, femoral and carotid atherosclerosis

To examine whether circulating levels of matrix metalloproteinase 9 (MMP-9) were associated with ultrasound-assessed intima-media thickness (IMT) and echolucent plaques in the carotid and femoral arteries. To examine preanalytical sources of variability in MMP-9 concentrations related to sampling procedures. Plasma and serum MMP-9 levels were compared with ultrasound assessed measures of femoral and carotid atherosclerosis, in a cross-sectional study of 61-year-old men (n = 473). Preanalytical sources of variability in MMP-9 levels were examined in 10 healthy subjects. Main outcome measures were circulating levels of MMP-9 in serum and plasma, IMT of the carotid and femoral arteries, and plaque status based on size and echolucency. Research unit at university hospital. Plasma concentrations of total and active MMP-9 were associated with femoral artery IMT independently of traditional cardiovascular risk factors, and were higher in subjects with moderate to large femoral plaques. Plasma MMP-9 concentration was higher in men with echolucent femoral plaques (P = 0.006) compared with subjects without femoral plaques. No similar associations were found for carotid plaques. MMP-9 concentrations were higher in serum than in plasma, and higher when sampling was performed with Vacutainer than with syringe. MMP-9 levels in serum were more strongly associated with peripheral neutrophil count compared with MMP-9 levels in plasma. Plasma MMP-9 levels were associated with atherosclerosis in the femoral artery, and total MMP-9 concentration was higher in men with echolucent femoral plaques. The choice of sample material and sampling method affect the measurements of circulating MMP-9 levels.

MMP-9 protein level does not reflect overall MMP activity in the airways of patients with COPD

Animal and human studies have implicated an imbalance of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in the pathogenesis of chronic obstructive pulmonary disease (COPD). MMP-9 protein is increased in COPD and we hypothesized that total MMP activity would be raised although this has not previously been measured. Using fluorescence and biotin labelled MMP assays, RT-PCR, western blotting and enzyme-linked immunosorbent assay we examined total MMP activity, specific gelatinase, elastase, collagenase activity, TIMP-1 and TIMP-2 in induced sputum from smokers with COPD and smokers without COPD. Induced sputum was obtained from 15 smokers with COPD and 14 smokers without COPD. MMP-9 levels were higher in those with COPD compared with controls (p<0.05). Total MMP activity, specific gelatinase, collagenase and elastase activities were not higher in COPD patients. In addition, reduced MMP activity was correlated with increasing airflow obstruction in COPD (p=0.016). MMP-9 protein but not MMP activity was higher in sputum of COPD patients compared with controls. These results suggest that MMP-9 levels may not reflect the overall MMP activity in the airways of patients with COPD suggesting a complex relationship between MMP-9 protein and activity. Further studies of MMPs in COPD should comprise activity measures in addition to protein levels.

The effects of eight-week walking programs of two different intensities on serum lipids and circulating markers of collagen remodelling in humans

Aim This study examined the effects of eight weeks of walking programs on serum lipids, circulation matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1). Methods Thirty-seven premenopausal women (30–55 years) completed eight weeks of walking programs either at moderate (∼5.25 ± 0.28 km/h; ∼53% maximum heart rate reserve (HRR max) or high-intensity (∼7.33 ± 0.24 km/h; ∼73% HRR max). Non-walking, sedentary women served as controls. Estimated V O 2 max was assessed with a 2 km walking test; body composition was measured using body composition analyzer; lipid, MMP-9, and TIMP-1 concentrations were measured in serum. Results After eight weeks, estimated V O 2 max improved in high-intensity walking group (HIWG; p < 0.01) and in moderate-intensity walking group (MIWG; p < 0.05); BMI, and percent body fat decreased ( p < 0.05) in both exercise groups. Only HIW caused significant reductions in total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), and MMP-9 levels ( p < 0.05); we found no significant changes in high-density lipoprotein-cholesterol and triglyceride levels in any of the groups. Exercise did not cause any significant changes in TIMP-1 levels apart from a nearly significant reduction in HIWG ( p = 0.084). Conclusion Walking programs with different intensity bring about favorable changes; but significant improvements for protective effects against cardiovascular diseases may be attained only with high-intensity walking programs due to the reduction in TC, LDL-C and MMP-9 levels.

Effect of collagen turnover and matrix metalloproteinase activity on healing of venous leg ulcers

The presence of fibrous tissue in poorly healing venous leg ulcers suggests abnormal collagen metabolism. The aim was to determine whether there were differences in collagen turnover and matrix metalloproteinase (MMP) activity between ulcers that healed, those that did not heal and normal skin. Biopsies were taken from the ulcers of 12 patients whose venous ulcers went on to heal and 15 patients whose ulcers failed to heal despite 12 months of compression bandaging. Biopsies were taken from 15 normal controls. Collagen turnover (collagen III N-terminal propeptide (PIIINP) and degraded collagen), and total MMP, MMP-1 and MMP-3 activities were measured. PIIINP and degraded collagen levels were higher in ulcers that healed compared with lesions that failed to heal (P = 0.005 and P < 0.001 respectively) and normal skin (P = 0.003 and P < 0.001). MMP-1 activity was also higher in healing ulcers than resistant ulcers (P < 0.001) and normal skin (P < 0.001). Significantly more total MMP activity was present in all ulcers than in normal skin (P < 0.001), but there was no difference in total MMP (and MMP-3 activity) between ulcers that healed and those that did not. Rapidly healing venous leg ulcers had increased collagen turnover and MMP-1 activity, which appeared to differentiate them from those that failed to heal within 12 months.

In vitro hypoxia increases production of matrix metalloproteinases and tryptase in isolated rat lung mast cells

Chronic hypoxia results in hypoxic pulmonary hypertension characterized by fibrotization and muscularization of the walls of peripheral pulmonary arteries. This vessel remodeling is accompanied by an increase in the amount of lung mast cells (LMC) and the presence of small collagen cleavage products in the vessel walls. We hypothesize that hypoxia activates LMC, which release matrix metalloproteinases (MMPs) cleaving collagen and starting increased turnover of connective tissue proteins. This study was designed to determine whether in vitro hypoxia stimulates production of MMPs in rat LMC and increases their collagenolytic activity. The LMC were separated on the Percoll gradient and then were divided into two groups and cultivated for 24 h in 21 % O(2) + 5 % CO(2) or in 10 % O(2) + 5 % CO(2). Presence of the rat interstitial tissue collagenase (MMP-13) in LMC was visualized by immunohistological staining and confirmed by Western blot analysis. Total MMPs activity and tryptase activity were measured in both cultivation media and cellular extracts. Exposure to hypoxia in vitro increased the amount of cells positively labeled by anti-MMP-13 antibody as well as activities of all measured enzymes. The results therefore support the concept that LMC are an important source of increased collagenolytic activity in chronic hypoxia.

A Biochemical Model of Matrix Metalloproteinase 9 Activation and Inhibition

Matrix metalloproteinases (MMPs) are a class of extracellular and membrane-bound proteases involved in an array of physiological processes, including angiogenesis. We present a detailed computational model of MMP9 activation and inhibition. Our model is validated to existing biochemical experimental data. We determine kinetic rate constants for the processes of MMP9 activation by MMP3, MMP10, MMP13, and trypsin; inhibition by the tissue inhibitors of metalloproteinases (TIMPs) 1 and 2; and MMP9 deactivation. This computational approach allows us to investigate discrepancies in our understanding of the interaction of MMP9 with TIMP1. Specifically, we find that inhibition due to a single binding event cannot describe MMP9 inhibition by TIMP1. Temporally accurate biphasic inhibition requires either an additional isomerization step or a second lower affinity isoform of MMP9. We also theoretically characterize the MMP3/TIMP2/pro-MMP9 and MMP3/TIMP1/pro-MMP9 systems. We speculate that these systems differ significantly in their time scales of activation and inhibition such that MMP9 is able to temporarily overshoot its final equilibrium value in the latter. Our numerical simulations suggest that the ability of pro-MMP9 to complex TIMP1 increases this overshoot. In all, our analysis serves as a summary of existing kinetic data for MMP9 and a foundation for future models utilizing MMP9 or other MMPs under physiologically well defined microenvironments.

Inhibition of integrative repair of the meniscus following acute exposure to interleukin‐1 in vitro

Damage or loss of the meniscus is associated with progressive osteoarthritic degeneration of the knee joint. Injured and degenerative joints are characterized by elevated levels of the pro-inflammatory cytokine interleukin-1 (IL-1), which with prolonged exposure can induce catabolic and anti-anabolic activities that inhibit tissue repair. We used an in vitro model system to examine the hypotheses that acute exposure to IL-1 inhibits meniscal repair, and that an IL-1-mediated increase in matrix metalloproteinase (MMP) activity is associated with the inhibition of repair. Integrative tissue repair was studied between concentric explants of porcine medial menisci that were treated with IL-1alpha acutely (100 pg/mL for 1 or 3 days) or chronically (100 pg/mL for entire culture duration). After 14 and 28 days in culture, biomechanical testing, cell viability, and histology were performed to assess meniscal repair. Total specific MMP activity in the culture media was measured using a quenched fluorescent substrate. As little as 1 day of IL-1 exposure significantly reduced shear strength, cell accumulation, and tissue repair compared to controls. IL-1 exposure for 1 or 3 days significantly increased MMP activity that subsided by day 9. With chronic IL-1 exposure, MMP activity remained elevated for the duration of culture and was negatively correlated with repair strength. Our study shows that short-term exposure to physiologically relevant concentrations of IL-1 significantly reduces meniscal repair in vitro, and thus may potentially inhibit the intrinsic repair response in vivo. The suppression of IL-1 or MMP expression and/or activity warrant investigation as potential strategies for promoting meniscal repair.

Relaxin stimulates MMP-2 and α-smooth muscle actin expression by human periodontal ligament cells

The main cells in the periodontal ligament (PDL) are the fibroblasts, which play an important role in periodontal remodelling. Matrix metalloproteinases (MMPs) are largely responsible for the degradation of extracellular matrix proteins in the PDL. Previous studies have indicated that MMP production can be stimulated by the hormone relaxin. This hormone facilitates delivery by softening the connective tissues of the reproductive tract, and it prepares the mammary gland for lactation. Periodontal remodelling takes place during orthodontic tooth movement, which might be enhanced by relaxin. Therefore, we investigated the effects of relaxin on gelatinase expression of human PDL cells. Cultures of human PDL cells were incubated with relaxin. Gelatinase (MMP-2 and -9) expression, α-smooth muscle actin expression (α-SMA), total MMP activity and DNA content were measured. Both proMMP-2 and active MMP-2 was identified in the cultures. There was a clear trend showing a dose-dependent increase of MMP-2 production, which was significant at 250 ng/ml. Total MMP activity was not affected. A stimulation of α-SMA expression was found at 50 ng/ml. The results indicate that relaxin activates human PDL cells by the stimulation of MMP-2 and α-smooth muscle actin.

A neurokinin‐1 receptor antagonist that reduces intraabdominal adhesion formation increases peritoneal matrix metalloproteinase activity

Adhesions remain a significant complication of abdominal surgery. There is a growing body of evidence suggesting that remodeling of peritoneal extracellular matrix by matrix metalloproteinases (MMPs) is involved in adhesion formation. We have shown that administration of a specific neurokinin-1 receptor (NK-1R) antagonist (CJ-12,255, Pfizer) to rats within 5 hours of surgery reduces intraabdominal adhesion formation. Because substance P (SP), the primary NK-1R ligand, is known to augment tissue fibrosis, the aim of this study was to determine the effects of NK-1R antagonist administration on peritoneal MMP expression and activity 24 hours after surgery in a rat adhesion model. Following laparotomy, four ischemic buttons were created on the peritoneum of rats that received either an intraperitoneal NK-1R antagonist or a vehicle at surgery. Adhesion formation was assessed 7 days later. Peritoneal fluid and tissue were collected at 24 hours to assess total MMP activity, as well as MMP-2, MMP-8, and MMP-9 activity. Specific MMP and tissue inhibitors of MMP mRNAs were measured, and the effects of SP on MMP-3 expression were determined in Met-5A cells, a human peritoneal mesothelial cell line. NK-1R antagonist administration reduced adhesion formation by 47% (p<0.05) at 7 days and significantly increased the total MMP activity in peritoneal fluid at 24 hours. There was an accompanying increase (p<0.05) in MMP-8 and MMP-9 mRNA expression and activity in peritoneal tissue and fluid, respectively. MMP-3 mRNA was also increased in the 24-hour peritoneal tissue, and exposure of Met-5A cells to SP reduced MMP-3 expression and activity. These data support a role for MMPs, specifically MMP-3, MMP-8, and MMP-9, in intraabdominal adhesion formation and suggest that the NK-1R antagonist may reduce adhesions, in part, by increasing MMP activity in the peritoneum by 24 hours after surgery.

Bronchoalveolar matrix metalloproteinase 9 relates to restrictive lung function impairment in systemic sclerosis

Systemic sclerosis (SSc) is frequently associated with interstitial lung disease (ILD) often leading to lung fibrosis. In this study we investigated whether matrix metalloproteinase 9 (MMP-9) and its natural inhibitor; the tissue inhibitor of matrix metalloproteinase 1 (TIMP-1), would be associated with remodelling in ILD in SSc. Levels of total MMP-9, pro-MMP-9 and TIMP-1 were measured in bronchoalveolar lavage (BAL) fluid from nine SSc patients with ILD, seven SSc patients without ILD and 16 age- and sex-matched healthy controls. Total MMP-9 and pro-MMP-9 levels were significantly elevated in SSc patients with ILD, compared to levels in SSc patients without ILD and healthy controls. In SSc patients with ILD calculated active MMP-9 levels were significantly higher than in SSc patients without ILD and tended to be higher than in healthy controls. TIMP-1 levels were elevated in both patient groups compared to healthy controls. Total-, pro- and active MMP-9 levels as well as pro-MMP-TIMP-1 and active MMP-9/TIMP-1 ratios were inversely associated with total lung capacity. The present study suggests that MMP-9 plays a pathophysiological role in the remodelling in ILD and lung fibrosis associated with SSc, and may represent a new therapeutic target in this condition.

Gelatinase concentrations and zymographic profiles in human breast cancer: Matrix metalloproteinases circulating in plasma are better markers for the subclassification and early prediction of cancer: The coagulation/fibrinolysis pathways alter the release, activation and recovery of different gelatinases in serum

Gelatinase concentrations and zymographic profiles in human breast cancer: Matrix metalloproteinases circulating in plasma are better markers for the subclassification and early prediction of cancer: The coagulation/fibrinolysis pathways alter the release, activation and recovery of different gelatinases in serum

Vascular Endothelial Growth Factor Induces Endothelin-1 Production via Matrix Metalloproteinase-2 Rather than Endothelin-Converting Enzyme-1

Objective: To investigate the mechanism of vascular endothelial growth factor (VEGF)-induced endothelin-1 production in human umbilical vein endothelial cells (HUVECs). Methods: Endothelin-1 levels were measured in conditioned medium of women with preeclampsia HUVECs were treated with different concentrations of VEGF165 and at various time intervals. Next, we measured endothelin-1 levels after HUVECs were also incubated with VEGF and endothelin-converting enzyme-1 (ECE-1) inhibitor or tissue inhibitors of matrix metalloproteinase-2 (TIMP-2). Additionally, the circulating levels of total and free VEGF, matrix metalloproteinase-2 (MMP-2), and endothelin-1 were measured in 20 preeclamptic patients and 20 healthy pregnant controls. Results: HUVECs treated with VEGF increased their endothelin-1 production in a concentration and time-dependent manner. The production of endothelin-1 was inhibited by TIMP-2, but not by the ECE-1 inhibitor. Total VEGF, MMP-2, and endothelin-1 concentrations were higher in preeclampsia and showed significant positive correlations between them. Conclusion: These findings suggest that VEGF-induced endothelin-1 production might be mediated by MMP-2 rather than by ECE-1 upregulation.

Collagen I and thrombin activate MMP-2 by MMP-14-dependent and -independent pathways: implications for airway smooth muscle migration

Increased proinflammatory mediators and ECM deposition are key features of the airways in asthma. Matrix metalloproteinases (MMPs) are produced by airway smooth muscle (ASM) cells and have multiple roles in inflammation and tissue remodeling. We hypothesized that components of the asthmatic airway would stimulate MMP production and activation by ASM and contribute to airway remodeling. We measured human ASM-derived MMP mRNA, protein, and activity by real-time RT-PCR, zymography, Western blotting, and MMP activity assay. Collagen I and thrombin caused a synergistic increase in MMP-2 protein and total MMP activity but paradoxically decreased MMP-2 mRNA. Additionally, collagen I activated MMP-2 in culture supernatants independent of the cell surface. Together, collagen I and thrombin strongly enhanced MMP-14 mRNA and protein but had no effect individually, suggesting increased MMP-14, the activating protease for MMP-2, may be partially responsible for MMP-2 activation. Furthermore, collagen I reduced tissue inhibitor of metalloproteinase-2 protein (TIMP-2). We examined the role of MMPs in functions of ASM related to airway remodeling and found migration and proliferation were MMP dependent, whereas adhesion and apoptosis were not. Ilomastat inhibited migration by 25%, which was also inhibited by TIMPs 1-4 and increased by the MMP-2 activator thrombin. These in vitro findings suggest that the environment within the airways of patients with asthma enhances MMP-2 and -14 protein and activity by a complex interaction of transcriptional and posttranscriptional mechanisms, which may contribute to ASM migration.

Disruption of an SP2/KLF6 Repression Complex by SHP Is Required for Farnesoid X Receptor-induced Endothelial Cell Migration

The farnesoid X receptor (FXR) signaling pathway regulates bile acid and cholesterol homeostasis. Here, we demonstrate, using a variety of gain- and loss-of-function approaches, a role of FXR in the process of cell motility, which involves the small heterodimeric partner (SHP)-dependent up-regulation of matrix metalloproteinase-9. We use this observation to reveal a transcriptional regulatory mechanism involving the SP/KLF transcription factors, SP2 and KLF6. Small interference RNA-based silencing studies in combination with promoter, gel shift, and chromatin immunoprecipitation assays indicate that SP2 and KLF6 bind to the matrix metalloproteinase-9 promoter and together function to maintain this gene in a silenced state. However, upon activation of FXR, SHP interacts with SP2 and KLF6, disrupting the SP2/KLF6 repressor complex. Thus, together, these studies identify a mechanism for antagonizing Sp/KLF protein repression function via SHP, with this process regulating endothelial cell motility.