Matrix Metallopeptidase 9 Activity Research Articles

Lonicera japonica Thunb. Ethanol Extract Exerts a Protective Effect on Normal Human Gastric Epithelial Cells by Modulating the Activity of Tumor-Necrosis-Factor-α-Induced Inflammatory Cyclooxygenase 2/Prostaglandin E2 and Matrix Metalloproteinase 9.

Gastric inflammation-related disorders are commonly observed digestive system illnesses characterized by the activation of proinflammatory cytokines, particularly tumor necrosis factor-α (TNF-α). This results in the induction of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PEG2) and matrix metallopeptidase-9 (MMP-9). These factors contribute to the pathogenesis of gastric inflammation disorders. We examined the preventive effects of Lonicera japonica Thunb. ethanol extract (Lj-EtOH) on gastric inflammation induced by TNF-α in normal human gastric mucosa epithelial cells (GES-1). The GES-1 cell line was used to establish a model that simulated the overexpression of COX-2/PGE2 and MMP-9 proteins induced by TNF-α to examine the anti-inflammatory properties of Lj extracts. The results indicated that Lj-EtOH exhibits significant inhibitory effects on COX-2/PEG2 and MMP-9 activity, attenuates cell migration, and provides protection against TNF-α-induced gastric inflammation. The protective effects of Lj-EtOH are associated with the modulation of COX-2/PEG2 and MMP-9 through the activation of TNFR-ERK 1/2 signaling pathways as well as the involvement of c-Fos and nuclear factor kappa B (NF-κB) signaling pathways. Based on our findings, Lj-EtOH exhibits a preventive effect on human gastric epithelial cells. Consequently, it may represent a novel treatment for the management of gastric inflammation.

Soluble CD147 regulates endostatin via its effects on the activities of MMP-9 and secreted proteasome 20S.

During progression of rheumatoid arthritis (RA), angiogenesis provides oxygen and nutrients for the cells' increased metabolic demands and number. To turn on angiogenesis, pro-angiogenic factors must outweigh anti-angiogenic factors. We have previously shown that CD147/extracellular matrix metalloproteinase inducer (EMMPRIN) can induce the expression of the pro-angiogenic factors vascular endothelial growth factor (VEGF) and matrix metallopeptidase 9 (MMP-9) in a co-culture of the human HT1080 fibrosarcoma and U937 monocytic-like cell lines. However, whether CD147 influences anti-angiogenic factors was not known. We now show that relative to single cultures, the co-culture of these cells not only enhanced pro-angiogenic factors but also decreased the anti-angiogenic factors endostatin and thrombospondin-1 (Tsp-1), generally increasing the angiogenic potential as measured by a wound assay. Using anti-CD147 antibody, CD147 small interfering RNA (siRNA), and recombinant CD147, we demonstrate that CD147 hormetically regulates the generation of endostatin but has no effect on Tsp-1. Since endostatin is cleaved from collagen XVIII (Col18A), we applied different protease inhibitors and established that MMP-9 and proteasome 20S, but not cathepsins, are responsible for endostatin generation. MMP-9 and proteasome 20S collaborate to synergistically enhance endostatin generation, and in a non-cellular system, CD147 enhanced MMP-9 activity and hormetically regulated proteasome 20S activity. Serum samples obtained from RA patients and healthy controls mostly corroborated these findings, indicating clinical relevance. Cumulatively, these findings suggest that secreted CD147 mediates a possibly allosteric effect on MMP-9 and proteasome 20S activities and can serve as a switch that turns angiogenesis on or off, depending on its ambient concentrations in the microenvironment.

Melatonin attenuates monocrotaline-induced hepatic sinusoidal obstruction syndrome in rats via activation of Sirtuin-3.

Melatonin possesses potent hepatoprotective properties, but it remains to be elucidated whether melatonin has a therapeutic effect on monocrotaline (MCT)-induced hepatic sinusoidal obstruction syndrome (HSOS). In this study, male Sprague Dawley rats were intraperitoneally injected with melatonin or the same volume of vehicle at 0 and 24 h after MCT intragastric administration. Next, hematoxylin-eosin staining and electron microscopy were performed to evaluate the hepatic sinusoidal injury of rats. Endothelial cell marker RECA-1 was observed by immunohistochemistry. Hepatic oxidative stress was analyzed by detecting malondialdehyde, glutathione S-transferase, and reactive oxygen species. Assessment of liver function was carried out byanalysis of serum aspartate aminotransferase, alanine aminotransferase, total bilirubin, and albumin levels. Real-time polymerase chain reaction and Western blot analysis were used to identifyliver Sirtuin-3 (SIRT3) and active matrix metallopeptidase 9 (MMP-9) expression. Besides, liver sinusoidal endothelial cells (LSECs) were used for the in vitro functional verification experiment. Specifically, liver histology of the melatonin-treated groups showed that the pathological damages caused by MCT were significantly attenuated, total HSOS scores were decreased, and the elevation of serum hyaluronic acid observed in the model group was also reduced. Moreover, melatonin treatment also improved the survival of rats after partial hepatectomy. Administration of melatonin ameliorated MCT-induced LSECs injury, hepatic oxidative stress, and hepatic dysfunction. Furthermore, melatonin treatment increased SIRT3 expression while attenuating MMP-9 activity in liver tissues. Cell experiment also demonstrated that SIRT3 might mediate the protective effect of melatonin on LSECs. Collectively, our study provided the potential rationale for the application of melatonin for the prevention of MCT-induced HSOS.

Evaluation of delphinidin as a storage medium for avulsed teeth

BackgroundDelphinidin (DP), an anthocyanidin found in blueberries, has antioxidant and anti-inflammatory effects. This study aimed to investigate the efficacy of DP as a storage medium for avulsed teeth.MethodsHuman periodontal ligament cells were cultured and exposed to DP solution (10, 50, and 100 μM), Dulbecco’s modified Eagle’s medium, Hank’s balanced salt solution and tap water. Cell counting kit-8 assays were performed after 0.5, 1, 6, and 24 h to measure the cell viability. Nitric oxide assays and gelatin zymography were performed to evaluate the anti-inflammatory effects of DP. Reverse transcription-polymerase chain reaction was used to determine the expression levels of inflammatory cytokines.ResultsThe viability of periodontal ligament cells was greatest at 100 μM DP. At 1 h, 100 μM DP decreased nitric oxide synthesis (p < .0167). Matrix metallopeptidase-9 activity was inhibited by DP in a dose-dependent manner (p < .0167). Moreover, treatment with 100 μM DP decreased the expression levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-8 in periodontal ligament cells (p < .0167).ConclusionsWithin the limits of this study, DP preserved the viability and suppressed the inflammatory response of periodontal ligament cells. These findings suggest that DP could be promising for preservation of avulsed teeth.

Downregulation of RAB7 and Caveolin-1 increases MMP-2 activity in renal tubular epithelial cells under hypoxic conditions.

Tubulointerstitial fibrosis leads to tubular basement membrane thickening and accumulation of interstitial extracellular matrix (ECM). Matrix metallopeptidase-2 (MMP-2) is involved in the breakdown of ECM. Chronic hypoxia often occurs in the kidney tissues of patients with chronic kidney disease. Our previous study reported the effect of autophagy and endocytosis on MMP-2 activity in hypoxia-treated HK-2 cells. In this study, the relationship between the Ras-related protein Rab-7a (RAB7) and MMP-2 activity was further investigated. RAB7 overexpression decreased MMP-2 activity. In contrast, the results for RAB7 knockdown displayed the opposite pattern. Short hairpin RNA technology was used to knockdown Caveolin-1 (Cav-1) or Beclin-1 (Bec-1) in HK-2 cells. The two genes displayed differential effects on MMP-2 activity. Cav-1 and RAB7 interference increased MMP-2 activity. This study suggested that autophagy and endocytosis, RAB7, Cav-1, and Bec-1 may serve as potential mediators for altered MMP-2 activity.

Hyperbaric oxygen therapy ameliorates the symptoms of post-concussion syndrome by inhibiting MMP-9 activity: a randomized controlled trial in Indonesia

Introduction: Post-concussion syndrome is common in young adults and can greatly interfere with the quality of daily life. It has a wide range of symptoms that require prompt and well-targeted treatment to avoid further brain impairment. Hyperbaric oxygen therapy (HBOT) is a promising regenerative treatment option for these patients to help prevent the progression of post-concussion syndrome. This study aims to determine whether HBOT accelerates the healing process and reduces symptoms in patients with post-concussion syndrome. Methods: 20 patients with post-concussion syndrome participated in this randomized controlled trial study. After receiving standard mild traumatic brain injury treatment in accordance with the Advanced Trauma Life Support guidelines, the patients were divided into HBOT and control groups. Matrix metallopeptidase 9 (MMP-9) levels and Rivermead Post-Concussion Symptoms Questionnaire (RPQ) scores were used to compare the two groups (before HBOT &amp; after 1st, 3rd, and 5th week). Results: The study sample was predominantly male (65%) with an average age of 60 years old. HBOT reduced serum MMP-9 levels by nearly 20 ng/mL (p &lt; 0.001) compared with the control treatment. The efficacy of HBOT was also reflected in the RPQ scores, which were significantly lower in the HBOT group than the control group (before HBOT &amp; after 1st, 3rd, 5th week) (-3.80 on RPQ-3, p = p&lt;0.001; -16.20 on RPQ-13, p = p&lt;0.001). Conclusion: HBOT ameliorated the symptoms associated with post-concussion syndrome through a mechanism that involves MMP-9 activity. The accelerated recovery observed in the present study supports the use of HBOT to treat post-concussion syndrome and potentially other forms of traumatic brain injury.

Apolipoprotein E isoforms differentially regulate matrix metallopeptidase 9 function in Alzheimer’s disease

Apolipoprotein E (APOE) has been shown to influence amyloid-β (Aβ) clearance from the brain in an isoform-specific manner. Our prior work showed that Aβ transit across the blood-brain-barrier was reduced by apoE4, compared to other apoE isoforms, due to elevated lipoprotein receptor shedding in brain endothelia. Recently, we demonstrated that matrix metallopeptidase 9 (MMP-9) induces lipoprotein receptor proteolysis in an apoE isoform-dependent manner, which impacts Aβ elimination from the brain. The current studies interrogated the relationship between apoE and MMP-9 and found that apoE impacted proMMP-9 cellular secretion from brain endothelia (apoE2 < apoE3 = apoE4). In a cell-free assay, apoE dose-dependently reduced MMP-9 activity, with apoE4 showing a significantly weaker ability to inhibit MMP-9 function than apoE2 or apoE3. Finally, we observed elevated MMP-9 expression and activity in the cerebrovasculature of both human and animal AD brain specimens with an APOE4 genotype. Collectively, these findings suggest a role for apoE in regulating MMP-9 disposition and may describe the effect of apoE4 on Aβ pathology in the AD brain.

Increased extracellular superoxide and MMP9 attenuated COMP stabilization of BMPR2 potentially participate in pulmonary hypertension development

The role of bone morphogenetic protein type 2 receptor (BMPR2) in the development of pulmonary hypertension was emphasized after identifying mutations of the gene encoding BMPR2 among patients with idiopathic pulmonary artery hypertension. Since then many studies were devoted to investigate restoring BMPR2 signaling as a therapeutic strategy. Also, roles for increased oxidant mechanisms have been identified in the lungs and right ventricle (RV) of several animal models of PH. However, it is unclear whether altered redox state plays a role in the regulation of the BMPR2 pathway in pulmonary vascular cells. Our current studies provide evidence for extracellular superoxide regulating BMPR2 expression via a Matrix Metallopeptidase 9 (MMP9)/cartilage oligomeric matrix protein (COMP) dependent pathway. Mice deficient of extracellular SOD (SOD3) show evidence of BMPR2 depletion both in the absence and presence of chronic hypoxia. Incubation of bovine pulmonary arteries under hypoxic conditions (1% O2 for 48h) increased MMP9 activity and expression which was associated with increased superoxide production and BMPR2 depletion. Moreover, adding exogenous superoxide dismutase (SOD) to the media inhibited MMP9 activation in response to acute hypoxia. In cultured human PASMC, 24 hours of hypoxia (1% O2) induced significant increase of MMP9 activity and reduced COMP expression. MMP9 contributes to COMP/BMPR2 depletion in chronic hypoxia based on MMP9 deficient mice showing a preserved expression of both COMP &amp; BMPR2 in lung tissues. Moreover, MMP9 deficient mice exposed to chronic hypoxia or Sugen/hypoxia showed a marked reduction of the elevated RV systolic pressures and Fulton index measurements of right ventricular hypertrophy compared to wild type mice under the same experimental conditions. ECHO measurements of PAAT/ET ratio, which correlate inversely and linearly with mean pulmonary artery pressure, in hypoxia‐exposed MMP9 KO mice showed increased ratios compared to animals exposed to chronic hypoxia or Sugen/hypoxia. These data are consistent with depletion of MMP9 reducing the development of pulmonary hypertension. Thus, increased extracellular superoxide appears to promote pathological loop of MMP9 mediated depletion of COMP leading to destabilization of BMPR2 and loss of its pulmonary hypertensive protective actions.Support or Funding InformationSupported by NIH Grants HL115124 &amp; HL129797

Characterizing the differential physiological effects of adipocytokines visfatin and resistin in liver cancer cells.

Background Obesity, a major public health concern, increases the risk of developing liver cancer which is the leading cause of cancer-related deaths worldwide. Obesity is associated with increased adiposity and macrophage infiltration both of which promote secretion of adipokines and cytokines in the tumor microenvironment. Specifically, visfatin and resistin have been detected at higher levels in the serum of obese individuals and liver tumors. However, the contribution of these adipocytokines in the progression of liver cancer remains unclear. Materials and methods The objective of this study was to characterize the effects of visfatin and resistin on HepG2, SNU-449 and HuH7 liver cancer cells. Cells exposed to visfatin and resistin were analyzed for fatty acid synthase protein, and phosphorylation of Akt and ERK tumorigenic signaling pathways, cell viability, lipogenesis, reactive oxygen species (ROS), matrix metallopeptidase 9 (MMP-9) enzyme activity and invasion. Results HepG2, SNU-449, and HuH7 liver cancer cells treated with visfatin and resistin increased cell viability, invasion, FASN protein, and Akt and ERK phosphorylation. Visfatin and resistin selectively increased ROS production in HepG2 and SNU-449 cells while there was no statistical difference in HuH7 cells. Visfatin and resistin stimulated lipogenesis in HepG2 cells while visfatin increased lipogenesis in SNU-449 cells, and visfatin nor resistin had an effect on lipogenesis in HuH7 cells. Lastly, visfatin and resistin increased MMP-9 enzyme activity in HepG2 and HuH-7 cells but only visfatin increased MMP-9 activity in SNU-449 cells. Conclusions Future studies are needed to determine if inhibition of ERK and Akt suppresses the visfatin and resistin-induced invasive liver cancer phenotype.

FURIN Inhibition Reduces Vascular Remodeling and Atherosclerotic Lesion Progression in Mice.

Objective- Atherosclerotic coronary artery disease is the leading cause of death worldwide, and current treatment options are insufficient. Using systems-level network cluster analyses on a large coronary artery disease case-control cohort, we previously identified PCSK3 (proprotein convertase subtilisin/kexin family member 3; FURIN) as a member of several coronary artery disease-associated pathways. Thus, our objective is to determine the role of FURIN in atherosclerosis. Approach and Results- In vitro, FURIN inhibitor treatment resulted in reduced monocyte migration and reduced macrophage and vascular endothelial cell inflammatory and cytokine gene expression. In vivo, administration of an irreversible inhibitor of FURIN, α-1-PDX (α1-antitrypsin Portland), to hyperlipidemic Ldlr-/- mice resulted in lower atherosclerotic lesion area and a specific reduction in severe lesions. Significantly lower lesional macrophage and collagen area, as well as systemic inflammatory markers, were observed. MMP2 (matrix metallopeptidase 2), an effector of endothelial function and atherosclerotic lesion progression, and a FURIN substrate was significantly reduced in the aorta of inhibitor-treated mice. To determine FURIN's role in vascular endothelial function, we administered α-1-PDX to Apoe-/- mice harboring a wire injury in the common carotid artery. We observed significantly decreased carotid intimal thickness and lower plaque cellularity, smooth muscle cell, macrophage, and inflammatory marker content, suggesting protection against vascular remodeling. Overexpression of FURIN in this model resulted in a significant 67% increase in intimal plaque thickness, confirming that FURIN levels directly correlate with atherosclerosis. Conclusions- We show that systemic inhibition of FURIN in mice decreases vascular remodeling and atherosclerosis. FURIN-mediated modulation of MMP2 activity may contribute to the atheroprotection observed in these mice.

Eurycoma longifolia, a promising suppressor of RANKL-induced differentiation and activation of osteoclasts: An in vitro mechanistic evaluation

BackgroundEurycoma longifolia (E. longifolia) has gained remarkable recognition due to its promising efficacy of stimulating bone formation in androgen-deficient osteoporosis. Numerous in vivo studies have explored the effects of E. longifolia on osteoporosis; however, the in vitro cellular mechanism was not discovered yet. ObjectivesThe present study was aimed to investigate the effect of E. longifolia on the proliferation, differentiation and maturation of osteoclasts and the translational mechanism of inhibition of osteoclastogenesis using RAW 264.7 cells as an in vitro osteoclastic model. Materials and methodsHaving assessed cytotoxicity, the cell viability, cell proliferation rate and osteoclastic differentiation capacity of E. longifolia was investigated by evaluating the tartrate-resistant acid phosphatase (TRAP) activity in receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclasts. Taken together, the time-mannered expression of osteoclast-related protein biomarkers such as matrix metallopeptidase-9 (MMP-9), cathepsin-K, TRAP, nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), superoxide (free radicals) generation and superoxide dismutase activity were also measured to comprehend the mechanism of osteoclastogenesis. ResultsE. longifolia did not show significant effects on cytotoxicity and cell proliferation of RAW 264.7 cells; however, a significant inhibition of cells differentiation and maturation of osteoclasts was observed. Moreover, a significant down-regulation of RANKL-induced TRAP activity and expression of MMP-9, cathepsin-K, TRAP, NFATc1 and generation of superoxide and enhanced superoxide dismutase activity was observed in E. longifolia treated cell cultures. ConclusionWe anticipated that E. longifolia that enhances bone regeneration on the one hand and suppresses osteoclast’s maturation on the other hand may have great therapeutic value in treating osteoporosis and other bone-erosive diseases such as rheumatoid arthritis and metastasis associated with bone loss.

Apolipoprotein E4 impairs spontaneous blood brain barrier repair following traumatic brain injury

BackgroundTraumatic Brain Injury (TBI) is a major cause of disability and mortality, to which there is currently no comprehensive treatment. Blood Brain Barrier (BBB) dysfunction is well documented in human TBI patients, yet the molecular mechanisms that underlie this neurovascular unit (NVU) pathology remains unclear. The apolipoprotein-E (apoE) protein has been implicated in controlling BBB integrity in an isoform dependent manner, via suppression of Cyclophilin A (CypA)–Matrix metallopeptidase-9 (MMP-9) signaling cascades, however the contribution of this pathway in TBI-induced BBB permeability is not fully investigated.MethodsWe exposed C57Bl/6 mice to controlled cortical impact and assessed NVU and BBB permeability responses up to 21 days post-injury. We pharmacologically probed the role of the CypA-MMP-9 pathway in BBB permeability after TBI using Cyclosporin A (CsA, 20 mg/kg). Finally, as the apoE4 protein is known to be functionally deficient compared to the apoE3 protein, we used humanized APOE mice as a clinically relevant model to study the role of apoE on BBB injury and repair after TBI.ResultsIn C57Bl/6 mice there was an inverse relationship between soluble apoE and BBB permeability, such that damaged BBB stabilizes as apoE levels increase in the days following TBI. TBI mice displayed acute pericyte loss, increased MMP-9 production and activity, and reduced tight-junction expression. Treatment with the CypA antagonist CsA in C57Bl/6 mice attenuates MMP-9 responses and enhances BBB repair after injury, demonstrating that MMP-9 plays an important role in the timing of spontaneous BBB repair after TBI. We also show that apoe mRNA is present in both astrocytes and pericytes after TBI.We report that APOE3 and APOE4 mice have similar acute BBB responses to TBI, but APOE3 mice display faster spontaneous BBB repair than APOE4 mice. Isolated microvessel analysis reveals delayed pericyte repopulation, augmented and sustained MMP-9 expression at the NVU, and impaired stabilization of Zonula Occludens-1, Occludin and Claudin-5 expression at tight junctions in APOE4 mice after TBI compared to APOE3 mice.ConclusionsThese data confirm apoE as an important modulator of spontaneous BBB stabilization following TBI, and highlights the APOE4 allele as a risk factor for poor outcome after TBI.

TPX2 silencing mediated by joint action of microvesicles and ultrasonic radiation inhibits the migration and invasion of SKOV3 cells.

Ovarian cancer, with its high morbidity, has one of the highest mortality rates among gynecological malignant tumors. Overexpression of targeting protein for Xklp2 (TPX2) has been identified in numerous malignant tumors. The present study sought to determine whether TPX2 silencing inhibited the growth and metastasis of ovarian cancer cells, and whether microvesicles- and ultrasonic radiation-mediated small interfering (si)RNA-TPX2 transfection may improve the therapeutic effect. The SKOV3 cell line, derived from papillary serous cytadenocarcinoma of the human ovary, was selected as a cell model. Cells were divided into five groups: Control, siRNA-TPX2, siRNA-TPX2 + microvesicle (M), siRNA-TPX2 + ultrasonic irradiation (UI), and siRNA-TPX2 + M + UI. Cell viability was evaluated under the aforementioned conditions via the Cell Counting kit 8 (CCK8) assay. Cell migration and invasion were detected using Transwell assays. The expression levels of associated genes, including epithelial cadherin (E-cadherin), metalloproteinase inhibitor 2 (TIMP-2), metastasis associated 1 (MTA1) and matrix metallopeptidase 2 (MMP2), were analyzed using reverse transcription-quantitative polymerase chain reaction analysis and western blotting. MMP2 activity was determined using a gelatin zymography assay. The results suggested that TPX2 serves an important role in the development of SKOV3 cells; it is additionally able to inhibit cell migration and invasion by upregulating E-cadherin and TIMP2, downregulating MMP2 and MTA1, and inhibiting the phosphorylation of p38 and c-Jun N-terminal kinase. The inhibitory effect of siRNA-TPX2 on SKOV3 cellular metastasis in the presence of microvesicles and ultrasonic radiation was observed to be improved compared with the control. It is proposed that the combination of microvesicles and ultrasonic radiation with TPX2 silencing has the potential to be an effective gene therapy against ovarian cancer.

Mito-Tempo prevents nicotine-induced exacerbation of ischemic brain damage.

Nicotine may contribute to the pathogenesis of cerebrovascular disease via the generation of reactive oxygen species (ROS). Overproduction of ROS leads to brain damage by intensifying postischemic inflammation. Our goal was to determine the effect of Mito-Tempo, a mitochondria-targeted antioxidant, on ischemic brain damage and postischemic inflammation during chronic exposure to nicotine. Male Sprague-Dawley rats were divided into four groups: control, nicotine, Mito-Tempo-treated control, and Mito-Tempo-treated nicotine. Nicotine (2 mg·kg-1·day-1) was administered via an osmotic minipump for 4 wk. Mito-Tempo (0.7 mg·kg-1·day-1 ip) was given for 7 days before cerebral ischemia. Transient focal cerebral ischemia was induced by occlusion of the middle cerebral artery for 2 h. Brain damage and inflammation were evaluated after 24 h of reperfusion by measuring infarct volume, expression of adhesion molecules, activity of matrix metalloproteinase, brain edema, microglial activation, and neutrophil infiltration. Nicotine exacerbated infarct volume and worsened neurological deficits. Nicotine did not alter baseline ICAM-1 expression, matrix metallopeptidase-2 activity, microglia activation, or neutrophil infiltration but increased these parameters after cerebral ischemia. Mito-Tempo did not have an effect in control rats but prevented the chronic nicotine-induced augmentation of ischemic brain damage and postischemic inflammation. We suggest that nicotine increases brain damage following cerebral ischemia via an increase in mitochondrial oxidative stress, which, in turn, contributes to postischemic inflammation. NEW & NOTEWORTHY Our findings have important implications for the understanding of mechanisms contributing to increased susceptibility of the brain to damage in smokers and users of nicotine-containing tobacco products.

Delayed formation of hematomas with ethanol preconditioning in experimental intracerebral hemorrhage rats.

Objective:Spontaneous intracerebral hemorrhage (ICH) accounts for 10%–15% of all strokes and causes high mortality and morbidity. In the previous study, we demonstrated that ethanol could aggravate the severity of brain injury after ICH by increasing neuroinflammation and oxidative stress. In this study, we further investigate the acute effects of ethanol on brain injury within 24 h after ICH.Materials and Methods:Totally, 66 male Sprague-Dawley rats were assigned randomly into two groups: saline pretreatment before ICH (saline + ICH), and ethanol pretreatment before ICH (ethanol + ICH). Normal saline (10 mL/kg) or ethanol (3 g/kg, in 10 mL/kg normal saline) was administered intraperitoneally 1 h before induction of experimental ICH. Bacterial collagenase VII-S (0.23 U in 1.0 μL sterile saline) was injected into the right striatum to induce ICH in the rats. We evaluated the hematoma expansion, hemodynamic parameters (heart rate and blood pressure), activated partial thromboplastin time (aPTT), prothrombin time (PT), and striatal matrix metallopeptidase 9 (MMP-9) expressions at 3, 6, 9, and 24 h after ICH.Results:The ethanol + ICH group exhibited decreased hematoma at 3 h after ICH; nevertheless, there was a larger hematoma compared with the saline + ICH group at 9 and 24 h after ICH. The ethanol + ICH group had lower blood pressure at 3, 6, and 9 h post-ICH, but both groups maintained similar heart rates after ICH. There was no significant difference in the aPTT and PT between the two groups. Incremental ethanol concentrations had no influence on collagenase VII-S activity at 120 min in vitro. MMP-9 expression was upregulated in the right striata of the ethanol + ICH group, especially at 3 and 9 h after ICH.Conclusion:Ethanol delayed hematoma formation in the first 3 h due to a hypotensive effect; however, the accelerated growth of hematomas after 9 h may be a sequela of ethanol-induced MMP-9 activation.

The Emerging Role of Zinc in the Pathogenesis of Multiple Sclerosis.

Our lab has previously demonstrated that multiple sclerosis-induced spinal cord white matter damage and motor deficits are mediated by the pathological disruption of zinc homeostasis. Abnormal vesicular zinc release and intracellular zinc accumulation may mediate several steps in the pathophysiological processes of multiple sclerosis (MS), such as matrix metallopeptidase 9 (MMP-9) activation, blood-brain barrier (BBB) disruption, and subsequent immune cell infiltration from peripheral systems. Oral administration of a zinc chelator decreased BBB disruption, immune cell infiltration, and spinal white matter myelin destruction. Therefore, we hypothesized that zinc released into the extracellular space during MS progression is involved in destruction of the myelin sheath in spinal cord white mater and in generation of motor deficits. To confirm our previous study, we employed zinc transporter 3 (ZnT3) knockout mice to test whether vesicular zinc depletion shows protective effects on multiple sclerosis-induced white matter damage and motor deficits. ZnT3 gene deletion profoundly reduced the daily clinical score of experimental autoimmune encephalomyelitis (EAE) by suppression of inflammation and demyelination in the spinal cord. ZnT3 gene deletion also remarkably inhibited formation of multiple sclerosis-associated aberrant synaptic zinc patches, MMP-9 activation, and BBB disruption. These two studies strongly support our hypothesis that zinc release from presynaptic terminals may be involved in multiple sclerosis pathogenesis. Further studies will no doubt continue to add mechanistic detail to this process and with luck, clarify how these observations may lead to development of novel therapeutic approaches for the treatment of multiple sclerosis.

Abstract 105: Association of Viscoelastic Material Properties and Extracellular Matrix Remodeling in Human Abdominal Aortic Aneurysmal Tissue

Objectives: Predicting rupture of abdominal aortic aneurysm (AAA) requires knowledge of both the rate of extracellular matrix (ECM) degradation and the pulsatile stress on the aortic tissue. The activity of matrix metallopeptidase 9 (MMP9) and its inhibitor, TIMP1, are associated with alterations in aortic ECM but it is unknown if these changes effect the dynamic viscoelastic properties. We hypothesize that increased levels of MMP9 within AAA tissue will be associated with a greater dynamic modulus (E*), as a surrogate of increased aortic wall stress. Methods: Human aneurysmal aortic tissue was obtained at the time of open AAA repair (n=11) and age-matched non-aneurysmal cadavers (n=10). Uniaxial viscoelastic material properties were measured in the circumferential orientation under physiologic preload (110 mmHg) and cyclic strain (± 5%@1Hz). Quantitative histologic and immunohistochemistry were preformed using Fiji imaging software. Aortic MMP9 and TIMP1 content and activity were quantified using western blot and zymography. Results: E* was greater (1862±464 vs 1362±405 kPa, p=0.02) in the AAA tissue as compared to non-aneurysmal tissue. AAA tissue contained less elastin (6.7±6.7 vs 23.4±8.7%, p=0.01) and a greater collagen/elastin ratio (19.9±20.6 vs 2.3±2.5%, p=0.05). Immunohistochemistry revealed 200% greater MMP9 content in the AAA tissue (Figure A &amp; B, 0.61 vs 0.03%, p=0.03). Increased MMP9 content was confirmed using a western blot (0.43 vs 0.06 AU, p&lt;0.01). No difference in relative MMP9 activity (4307 vs 2324 AU, p=0.25) or level of TIMP1 (0.03 vs 0.02, p=0.6) were observed. There was a positive linear correlation (Figure C, r 2 =0.47) between E* and MMP9 as determined by quantitative immunohistochemistry. Conclusions: Our data suggests a positive relationship between E* and MMP9 content. Increased tissue stiffness may trigger MMP9 production resulting in a positive-feedback loop, progressively increasing aortic wall stress and rupture risk.

Critical role of matrix metallopeptidase 9 in postoperative cognitive dysfunction and age-dependent cognitive decline.

BackgroundPostoperative cognitive dysfunction (POCD) is a significant clinical syndrome. Neuroinflammation is an important pathological process for POCD. However, it is not clear how systemic inflammation induced by surgery on peripheral tissues or organs is transmitted into the brain. We determined whether matrix metallopeptidase 9 (MMP9), a protein that can increase blood-brain barrier permeability, is critical in this transmission. The role of MMP9 in age-dependent cognitive decline was also determined.MethodsTwo-month old male C57BL/6J wild-type mice and MMP9-/- mice were randomly assigned to control or surgery groups. The surgery was right carotid artery exposure under isoflurane anesthesia. Cognitive function was tested from one week after the surgery by Barnes maze and fear conditioning. Cognitive function of 2-month old C57BL/6J mice was compared with that of 18-month old mice.ResultsSurgery increased the expression of interleukin 1β, interleukin 6 and ionized calcium binding adapter molecule 1, inflammation indicators, in the brain of the wild-type mice. Blood-brain barrier permeability was increased by surgery. Surgery also impaired the learning and memory of these mice. These surgical effects were absent in the MMP9-/- mice. Eighteen-month old wild-type mice had poorer performance in Barnes maze and fear conditioning tests and lower MMP9 protein expression and activity than did the 2-month old mice.ConclusionMMP9 is critical for transmission of systemic inflammation into the brain for POCD. MMP9 may also play a role in age-dependent cognitive decline.

M2-like macrophages induce colon cancer cell invasion via matrix metalloproteinases.

The inflammatory milieu plays an important role in colon cancer development and progression. Previously, we have shown that tumor-associated macrophages (TAMs), an important component of the tumor microenvironment, are enriched in tumors compared with normal tissue and confer a poorer prognosis. In the present study, we found that matrix metallopeptidase-9 (MMP-9), which degrades extracellular matrix proteins, was increased in biopsies from colon cancer patients and in mouse xenografts with SW480 cell-derived tumors. SW480 colon cancer cells exposed to M2-like macrophage-conditioned medium (M2-medium) exhibited increased MMP-9 mRNA, protein expression and gelatinase activity. A similar effect was obtained by the addition of tumor necrosis factor-α (TNFα) and leukotriene D4 (LTD4 ). MMP-9 expression and activity were reduced by a TNFα blocking antibody adalimumab and a cysteinyl leukotriene receptor 1 (CysLTR1, the receptor for LTD4 ) antagonist montelukast. M2-medium also induced changes in the epithelial-mesenchymal transition (EMT) markers E-cadherin, β-catenin, vimentin, and snail in SW480 cells. We also found that both M2-medium and TNFα and LTD4 induced stabilization/nuclear translocation of β-catenin. Furthermore, we also observed an elongated phenotype that may indicate increased invasiveness, as confirmed in a collagen I invasion assay. M2-medium increased the invasive ability, and a similar effect was also obtained by the addition of TNFα and LTD4 . The specific MMP inhibitor I or adalimumab and montelukast reduced the number of invasive cells. In conclusion, our findings show that M2-medium enriched in TNFα and LTD4 promote colon cancer cell invasion via MMP-9 expression and activation and the induction of EMT.

Methane ameliorates spinal cord ischemia-reperfusion injury in rats: Antioxidant, anti-inflammatory and anti-apoptotic activity mediated by Nrf2 activation

Methane is reported to have antioxidant, anti-inflammatory and anti-apoptotic properties. We investigated the potential neuroprotective effects of methane-rich saline (MS) on spinal cord ischemia-reperfusion injury and determined that its therapeutic benefits are associated with the activation of nuclear factor erythroid 2-related factor 2 (Nrf2). Rats received 9min of spinal cord ischemia induced by occlusion of the descending thoracic aorta plus systemic hypotension followed by a single MS treatment (10ml/kg, ip) and 72h reperfusion. MS treatment attenuated motor sensory deficits and produced high concentrations of methane in spinal cords during reperfusion, which increased Nrf2 expression and transcriptional activity in neurons, microglia and astrocytes in the ventral, intermediate and dorsal gray matter of lumbar segments. Heme oxygenase-1, superoxide dismutase, catalase and glutathione were upregulated; and glutathione disulfide, superoxide, hydrogen peroxide, malondialdehyde, 8-hydroxy-2-deoxyguanosine and 3-nitrotyrosine were downregulated in MS-treated spinal cords. MS treatment reduced neuronal apoptosis in gray matter zones, which was consistent with the suppression of cytochrome c release to the cytosol from the mitochondria and the activation of caspase-9 and -3. Throughout the gray matter, the activation of microglia and astrocytes was inhibited; the nuclear accumulation of phosphorylated nuclear factor-kappa B p65 was reduced; and tumor necrosis factor α, interleukin 1β, chemokine (C-X-C motif) ligand 1, intercellular adhesion molecule 1 and myeloperoxidase were decreased. MS treatment attenuated blood-spinal cord barrier dysfunction by preventing the expression and activity of matrix metallopeptidase-9 and disrupting tight junction proteins. Consecutive intrathecal injection of specific siRNAs targeting Nrf2 at 24-h intervals 3 days before ischemia reduced the beneficial effects of MS. Our data indicate that MS treatment prevents IR-induced spinal cord damage via antioxidant, anti-inflammatory and anti-apoptotic activities that involve the activation of Nrf2 signaling. Thus, methane may serve as a novel promising therapeutic agent for treating ischemic spinal cord injury.