Upregulation of MMP-9/TIMP-1 enzymatic system in eosinophilic meningitis caused by Angiostrongylus cantonensis

Proteolysis depends on the balance between the proteases and their inhibitors. Matrix metalloproteinase-9 (MMP-9) and its specific inhibitors, tissue inhibitors of metalloproteinases (TIMP), contribute to eosinophilic inflammatory reaction in the subarachnoid space of the Angiostrongylus cantonensis-infected mice. The expression of MMP-9 in cerebrospinal fluid (CSF) was significantly increased in mice with eosinophilic meningitis, compared to that in uninfected ones. However, the TIMP-1 levels were unchanged and remained at basal levels at all time points, even in uninfected mice. Elevated MMP-9 mRNA expression coincided with protein levels and proteolytic activity, as demonstrated by means of positive immunoreactivity and gelatin zymography. CSF protein contents correlated significantly with MMP-9 intensity and CSF eosinophilia. In addition, immunohistochemistry demonstrated MMP-9 and TIMP-1 localization in eosinophils and macrophages. When the specific MMP inhibitor, GM6001, was added, MMP-9 enzyme activity was reduced by 45.4%. The percentage of eosinophil increased significantly upon the establishment of infection, but subsided upon inhibition. These results show that MMP-9/TIMP-1 imbalance in angiostrongyliasis may be associated with eosinophilic meningitis.     

Association of matrix metalloproteinase-9 in eosinophilic meningitis of BALB/c mice caused by Angiostrongylus cantonensis

Induction of gelatinase in eosinophilic meningitis of BALB/c-strain mice was caused by Angiostrongylus cantonensis. Time-course studies showed that the molecular weight of 94-kDa gelatinase was detected at day 10 post-inoculation (PI), and reached a high intensity from days 15 to 25 PI. The 94-kDa gelatinase activity was clearly inhibited by EDTA and 1,10-phenanthroline, but not by leupeptin and phenylmethanesulphonyl fluoride. When immunoblots were performed using specific antiserums against the 94-kDa gelatinase B (matrix metalloproteinase-9; MMP-9) with cerebrospinal fluid (CSF), the 94-kDa immunopositive band was MMP-9. Immunohistochemistry studies demonstrated MMP-9 localisation within eosinophils and macrophages. The increased MMP-9 activity was closely associated with the rapid rise of CSF eosinophils, and the inflammatory reaction of the subarachnoid space. In contrast to changes in MMP-9, MMP-2 activity was constitutive and unaffected in this parasitic meningitis. These results show that MMP-9 was associated with eosinophilic meningitis, and that the enzyme may be a useful marker for angiostrongyliasis meningitis.     

Increased TIMP/MMP ratio in varicose veins: a possible explanation for extracellular matrix accumulation

Primary varicose veins are functionally characterized by venous back-flow and blood stagnation in the upright position. Dilatation and tortuosity provide evidence for progressive venous wall remodelling, with disturbance of smooth muscle cell/extracellular matrix organization. Affected areas are not uniformly distributed, some areas being hypertrophic, whereas others are atrophic or unaffected. In 12 varicose veins and ten control veins, the proteolytic enzyme/inhibitor balance which may participate in the remodelling of the venous wall was investigated. For this purpose, the presence and enzymatic activity of matrix metalloproteinases (MMP-2, MMP-9), tissue inhibitors of MMPs (TIMP-1, TIMP-2), urokinase-type (uPA) and tissue-type (tPA) plasminogen activators (PAs), and plasminogen activator inhibitor-1 (PAI-1) were quantified by western blot and gelatin or plasminogen-casein zymography. In addition, MMP-2, TIMP-1, TIMP-2, and PAI-1 levels were measured by ELISA. A high TIMP-1 level and a low MMP-2 level/activity were found in varicose veins (p<0.005), resulting in a three-fold increase in the TIMP-1/MMP-2 ratio in varicose versus control veins. Levels of PAs (uPA and tPA) as well as PAI-1 were both lower in varicose veins (p<0.005), with minimal change in the PAI/PA ratio. These results demonstrate that varicose veins are characterized by a higher than normal TIMP/MMP ratio, which may facilitate extracellular matrix accumulation in the diseased venous wall.     

Cerebrospinal fluid activity of tissue plasminogen activator in patients with neurological diseases.

AIM: To study cerebrospinal fluid (CSF) activity of tissue plasminogen activator (tPA) in patients with neurological diseases. METHODS: CSF tPA and urokinase (uPA) activities were studied using an immunocapture assay and zymography in 44 patients with neurological disease and 20 reference subjects. The patient group comprised three patients with meningitis, 21 with encephalitis, nine with acute lymphoblastic (n = 7) and myeloid (n = 2) leukaemia, seven with multiple sclerosis, three with facial paresis, and one with polyradiculitis. RESULTS: Raised tPA activities were observed in patients with multiple sclerosis, leukaemia and encephalitis. In contrast, there were no differences in the mean activities of tPA in patients with meningitis or other diseases compared with the reference subjects. The highest tPA activities were found in patients with multiple sclerosis. The mean activity in patients with leukaemia was higher than in those with meningitis and polyradiculitis, but not encephalitis and facial paresis. Although the CSF tPA activity correlated positively with age in reference subjects, no correlation was observed in patients. Samples were qualitatively screened for both tPA and uPA activity by zymography and positive samples were quantitated. Some of the samples had quantifiable levels of uPA activity: three of seven multiple sclerosis samples, 10 of 21 samples from patients with encephalitis and five of nine leukaemic samples. The highest activities were recorded in patients with leukaemia. uPA was not detected in the CSF of the patients with meningitis, facial paresis or polyradiculitis. CONCLUSIONS: Plasminogen activator activity can be measured reliably in CSF and the assessment of tPA activity may be useful for studying the pathogenesis of neurological diseases.     

A role of fibrinolytic activity in angiogenesis. Quantitative assay using in vitro method

The functional role of the fibrinolytic system in capillary growth was investigated using bovine capillary endothelial cells (BCEs) cultured on a Type I collagen gel matrix, into which the cells migrated to form capillary-like tubular structures. The length of the tubes formed were measured morphometrically using an image analyzer in the absence and presence of fibrinolytic proteases, namely plasminogen, plasminogen activators (PAs) and PA inhibitor (PAI). The addition of plasminogen (25 micrograms/ml) to the gel matrix significantly increased the length of BCE tubes found on the 9th day of culture (p less than 0.01), with a dose-dependent tendency. The simultaneous addition of a basic fibroblast growth factor (bFGF, 10 ng/ml) enhanced this tube formation as early as the 3rd day of culture (p less than 0.01). Cultured BCEs secreted both tissue-type and urokinase-type PAs (tPA and uPA) and PAI-1 into the culture medium, and the secretion of both PAs was enhanced by the addition of bFGF. However, the secreted tPA was composed mostly of an inactive form of tPA.PAI-1 complex, and the PA activity was derived mostly from uPA. Inhibitors of plasmin suppressed the enhancing effect of plasminogen on angiogenesis. In addition, anti-uPA IgG markedly inhibited the enhancing effect of plasminogen on the 4th and 7th days of culture (p less than 0.01), whereas anti-tPA IgG showed an inhibitory tendency only on the 4th day of culture (p less than 0.05). These findings indicate that the plasminogen-PAs system, especially uPA synthesized and secreted by BCEs, plays an important role in regulating angiogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

结核性脑膜炎患者脑脊液基质金属蛋白酶-9的检测意义

在脑膜炎的发展过程中,基质金属蛋白酶-9(MMP-9)对降解基底膜和损伤血脑屏障发挥重要作用,结核分枝杆菌能够刺激单核-巨噬细胞过度表达MMP-9.为检测结核性脑膜炎患者脑脊液MMP-9水平,评价其对结核性脑膜炎诊断及治疗的意义,应用ELISA方法检测12例结核性脑膜炎和9例病毒性脑膜炎患者脑脊液MMP-9水平,并选取5例非肿瘤非感染性头痛患者脑脊液作对照组.结果显示,结核性脑膜炎脑脊液MMP-9的水平显著增高,中枢神经系统并发症发生率与MMP-9的水平相关.结论为脑脊液MMP-9水平对结核性脑膜炎的诊断和预后有参考价值.     

Cellular and extracellular plasminogen activator and inhibitor in an experimental tumour.

We determined plasminogen activator (PA) and PA inhibitor (PAI) activities in the intra- and extracellular compartments of an experimental pancreatic ascites tumour with indirect and direct functional assays, and partially characterized these activities on SDS-polyacrylamide gels coupled with fibrin and reverse fibrin autography. Intact tumour cells caused lysis of plasminogen-rich but not plasminogen-free fibrin clots, and the extent of lysis of the former was related to tumour cell count. Direct assay of PA with a synthetic substrate yielded an equivalent of 109 urokinase units per 10(9) tumour cells. No PAI activity was demonstrated in tumour cells with functional assays. Contrary to tumour cells, cell-free ascitic fluids caused no lysis of fibrin clots. Instead, it inhibited tumour cell- and urokinase-induced, but not plasmin-induced, clot lysis in a dose-dependent fashion. Although functional assays failed to demonstrate PA in ascitic fluid and PAI in tumour cells, both activities were detected in electrophoresed samples of cell lysates and fluids by fibrin and reverse fibrin autography. In tumour cells, a mixture of tissue-type PA (tPA) and urokinase-type PA (uPA) were present. In the fluid, uPA together with two other PAs with greater molecular weights than tPA were detected.     

Cellular and extracellular plasminogen activator and inhibitor in an experimental tumour

We determined plasminogen activator (PA) and PA inhibitor (PAI) activities in the intra- and extracellular compartments of an experimental pancreatic ascites tumour with indirect and direct functional assays, and partially characterized these activities on SDS-polyacrylamide gels coupled with fibrin and reverse fibrin autography. Intact tumour cells caused lysis of plasminogen-rich but not plasminogen-free fibrin clots, and the extent of lysis of the former was related to tumour cell count. Direct assay of PA with a synthetic substrate yielded an equivalent of 109 urokinase units per 10(9) tumour cells. No PAI activity was demonstrated in tumour cells with functional assays. Contrary to tumour cells, cell-free ascitic fluids caused no lysis of fibrin clots. Instead, it inhibited tumour cell- and urokinase-induced, but not plasmin-induced, clot lysis in a dose-dependent fashion. Although functional assays failed to demonstrate PA in ascitic fluid and PAI in tumour cells, both activities were detected in electrophoresed samples of cell lysates and fluids by fibrin and reverse fibrin autography. In tumour cells, a mixture of tissue-type PA (tPA) and urokinase-type PA (uPA) were present. In the fluid, uPA together with two other PAs with greater molecular weights than tPA were detected.     

Therapeutic approaches to vascular protection in ischemic stroke

Reperfusion with recombinant tissue plasminogen activator (tPA) sometimes causes catastrophic hemorrhagic transformation (HT) in the ischemic brain. Consequently, the application of tPA has been strictly limited. Recent studies have indicated that matrix metalloproteinases (MMPs), especially MMP-9, play a critical role in blood brain barrier (BBB) disruption in the ischemic brain, leading to brain edema and HT. In the ischemic brain, free radicals and exogenous tPA itself can trigger MMP-9 activation through several signaling pathways containing LDL receptor-related protein (LRP) and proteinase-activated receptor 1 (PAR1). Therapeutic targeting of free radicals and MMP-9/t-PA related signaling pathways might be promising approaches to minimizing catastrophic HT in acute stroke patients. We provide an overview of the available scientific reports to improve our understanding of the mechanisms leading to HT, and highlight recent progress in the development of new therapeutic strategies for preventing HT in the post-stroke brain.     

Matrix metalloproteinase-9 knockout confers resistance to corneal epithelial barrier disruption in experimental dry eye

Altered corneal epithelial barrier function is the cause for ocular irritation and visual morbidity in dry eye disease. Increased matrix metalloproteinase (MMP)-9 activity has been observed in the tear fluid of dry eye patients. To determine the pathogenic role of MMP-9 in the corneal epithelial disease of dry eye, the effects of experimentally induced dry eye on corneal epithelial morphology and barrier function were compared in MMP-9 knockout mice and their wild-type littermates. Dry eye was created through cholinergic blockade and exposure to a desiccating environment. The tear fluid MMP-9 concentration increased in response to dryness in wild-type mice. Corneal epithelial permeability to three different-sized molecules increased in dry eye wild-type mice, but not in MMP-9 knockout mice. Topical administration of active MMP-9 to dry eye MMP-9 knockout mice significantly increased corneal epithelial permeability. Compared to MMP-9 knockout mice, wild-type mice showed greater desquamation of differentiated apical corneal epithelial cells that expressed the tight junction protein occludin in response to dryness. This was accompanied by an increase in lower sized (50 kd) occludin in the corneal epithelia of wild-type mice. These findings could be replicated in cultured human corneal epithelial cells that were treated with active MMP-9. These studies indicate that increased MMP-9 activity on the ocular surface in response to dryness disrupts corneal epithelial barrier function. This appears to be because of accelerated loss of tight junction bearing superficial corneal epithelial cells, perhaps by proteolytic cleavage of occludin.     

Reduction of hippocampal cell death and proteolytic responses in tissue plasminogen activator knockout mice after transient global cerebral ischemia

Knockout mice deficient in tissue plasminogen activator (tPA) are protected against hippocampal excitotoxicity. But it is unknown whether similar neuroprotection occurs after transient global cerebral ischemia, which is known to selectively affect the hippocampus. In this study, we tested the hypothesis that hippocampal cell death in tPA knockout mice would be reduced after transient global cerebral ischemia, and this neuroprotection would occur concomitantly with amelioration of both intra- and extracellular proteolytic cascades. Wild-type and tPA knockout mice were subjected to 20 min of transient bilateral occlusions of the common carotid arteries. Three days later, Nissl and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling staining demonstrated that hippocampal cell death was significantly reduced in tPA knockout brains compared with wild-type brains. Caspase-3 and the two major brain gelatinases (matrix metalloproteinase (MMP)-9 and MMP-2) were assessed as representative measurements of intra- and extracellular proteolysis. Post-ischemic levels of caspase-3, MMP-9 and MMP-2 were similarly reduced in tPA knockouts compared with wild-type hippocampi. Taken together, these data suggest that endogenous tPA contributes to hippocampal injury after cerebral ischemia, and these pathophysiologic pathways may involve links to aberrant activation of caspases and MMPs.     

Differential effect of p47phox and gp91phox deficiency on the course of Pneumococcal Meningitis

Bacterial meningitis is a severe inflammatory disease of the central nervous system and is characterized by massive infiltration of granulocytes into the cerebrospinal fluid (CSF). To assess the role of NADPH oxidase-derived reactive oxygen species (ROS) in pneumococcal meningitis, mice deficient in either the gp91 subunit (essential for functioning of the phagocyte enzyme) or the p47 subunit (essential for functioning of homologous enzymes in nonphagocytic cells) were intracisternally infected with live Streptococcus pneumoniae, and defined disease parameters were measured during the acute stage of infection. While none of the parameters measured (including CSF bacterial titers) were significantly different in gp91(-/-) and wild-type mice, the infection in p47(-/-) mice was associated with significantly increased inflammation of the subarachnoid and ventricular space, disruption of the blood-brain barrier, and the presence of interleukin-1 beta, tumor necrosis factor alpha, and matrix metalloproteinase 9 in the cortex. These changes were associated with approximately 10-fold-higher CSF bacterial titers in p47(-/-) mice than in wild-type mice (P < 0.001). In contrast to infection with live bacteria, the inflammatory response, including CSF leukocytosis, was significantly attenuated in p47(-/-) mice (but not gp91(-/-) mice) challenged with a fixed number of heat-inactivated pneumococci. Impairment of the host defense appeared to be responsible for the higher bacterial titers in p47(-/-) mice. Therefore, these results indicate that ROS generated by a gp91-independent NADPH oxidase(s) are important for establishing an adequate inflammatory response to pneumococcal CSF infection.     

Cerebrospinal fluid plasminogen activator inhibitor-1 in patients with neurological disease.

AIM: To study cerebrospinal fluid (CSF) concentrations of plasminogen activator inhibitor type-1 (PAI-1) in patients with neurological disease. METHODS: CSF PAI-1 concentrations were measured in 51 patients with neurological disease and 20 reference subjects using an ELISA. The patient group comprised three patients with viral meningitis, 20 with encephalitis, nine with acute lymphoblastic (n = 7) and myeloid (n = 2) leukaemia (with central nervous system involvement), and 19 with multiple sclerosis. RESULTS: Raised PAI-1 concentrations were observed in patients with leukaemia, encephalitis and multiple sclerosis. There was no difference in the mean concentrations of PAI-1 in patients with meningitis when compared with the reference subjects. The highest mean (SEM) PAI-1 concentration was found in patients with leukaemia (1.28 (0.36) ng/ml), and the next highest in those with encephalitis (1.19 (0.20) ng/ml). these values were much higher than those in patients with viral meningitis. In a previous report, raised CSF tissue-type plasminogen activator (tPA) activities were detected in patients with multiple sclerosis, leukaemia and encephalitis, with mean activities in decreasing order. PAI-1 concentrations in the same patients were the reverse of their corresponding tPA activities, being higher in those with leukaemia and encephalitis, than in patients with multiple sclerosis. There was no association between CSF PAI-1 concentrations and age in either patients or controls. Similarly, there was no association between CSF PAI-1 concentrations and urokinase-type plasminogen activator (uPA). CONCLUSIONS: Raised CSF PAI-1 concentrations may be used as a non-specific marker of neurological disease. Moreover, PAI-1 may play an important role in regulating the functions tPA, and probably uPA, in CSF.     

The p75NTR metastasis suppressor inhibits urokinase plasminogen activator, matrix metalloproteinase-2 and matrix metalloproteinase-9 in PC-3 prostate cancer cells

The p75 neurotrophin receptor (p75^NTR) has been characterized as a metastasis and tumor suppressor in prostate cancer. In order to investigate the mechanism(s) by which the p75^NTR functions as a metastasis suppressor in prostate cancer cells, we characterized the ectopic expression of p75^NTR on the urokinase plasminogen activator (uPA) and the type IV collagen matrix metalloproteinases (MMP-2 and MMP-9) in PC-3 human prostate cancer cells. Rank-order expression of p75^NTR greatly reduced protein levels and enzymatic activities of uPA, MMP-2, and MMP-9 as shown by immunoblot and zymography analyses. Conversely, expression of the MMP-9 antagonist, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) exhibited an increase in protein levels with an increase in p75^NTR levels, whereas TIMP-2 was not detected. Transient transfection with an inducible dominant negative antagonist Δp75^NTR rescued uPA, MMP-2, and MMP-9 protein levels and protease activities, and conversely suppressed TIMP-1 levels. Since p75^NTR signal transduction occurs via the NFκB and JNK pathways, antagonism of signaling intermediates in these pathways, using dominant negative IKKβ or dominant negative MKK-4, respectively, was shown to further decrease expression of uPA, MMP-2, and MMP-9 protein and enzymatic activity levels, and conversely up-regulate levels of TIMP-1. These results indicate that expression of uPA, MMP-2, MMP-9, and TIMP-1 are directly regulated by expression of p75^NTR and its downstream signal transduction cascade. These results suggest that the metastasis suppressor activity of p75^NTR is mediated, in part, by down-regulation of specific proteases (uPA, type IV collagenases) implicated in cell migration and metastasis.     

Induction of matrix metalloproteinase,cytokines and chemokines in rat cortical astrocytes exposed to plasminogen activators

Plasminogen activators are used in thrombolytic stroke therapy. However, it is increasingly recognized that they have other actions besides fibrinolysis. In this study, we assess potential pro-inflammatory effects of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) in rat cortical astrocytes. Both uPA and tPA induced rapid dose-dependent upregulation in MMP-2 and MMP-9, as demonstrated by zymography of conditioned media. In addition, a multiplex ELISA array demonstrated that patterned responses in chemokines and cytokines were also evoked. Exposure to tPA induced elevations in secreted MIP-2, MCP-1 and GRO/KC. Exposure to uPA induced elevations in secreted IFN-γ, TNF-α, GMCSF, MIP-1α, MIP-2, MIP-3α, MCP-1, RANTES and fractalkine. These data suggest that plasminogen activators may trigger selected pro-inflammatory responses at the neurovascular interface. Whether these effects influence thrombolytic stroke therapy warrants further investigation.     

Upregulation of prostaglandin E2 and matrix metalloproteinase 9 production by human macrophage-like cells: synergistic effect of capsular material and cell wall from Streptococcus suis

Streptococcus suis, an early colonizer of the upper respiratory tract, is a major swine pathogen worldwide that can cause meningitis, arthritis, pneumonia, and septicemia. While numerous studies on potential virulence factors of S. suis have been carried out over the past decade, the exact mechanisms by which this bacterium invades the host and migrates through the blood brain barrier (BBB) remain unclear. In the study presented here, we show that whole cells of S. suis were able to upregulate the production of prostaglandin E2 (PGE2) and matrix metalloproteinase 9 (MMP-9) by U937 human monoblastic cells differentiated into adherent macrophages in a dose- and time-dependent way. Capsular material and cell wall of S. suis were tested to determine which component was responsible for the induction of PGE2 and MMP-9 production by macrophages. The capsular material, even at low concentrations, strongly stimulated the production of PGE2 and MMP-9. While no stimulation was observed with the purified cell wall material, combining it with the capsular material resulted in a significant synergic effect on PGE2 and MMP-9 production. S. suis-mediated MMP-9 and PGE2 production by human macrophages may play a critical role in BBB disruption and tissue destruction.