Matrix Metallopeptidase 9 Activity Research Articles

Neuroprotective Effects of Valproic Acid on Blood-Brain Barrier Disruption and Apoptosis-Related Early Brain Injury in Rats Subjected to Subarachnoid Hemorrhage Are Modulated by Heat Shock Protein 70/Matrix Metalloproteinases and Heat Shock Protein 70/AKT Pathways.

Blood-brain barrier (BBB) disruption and neural apoptosis are thought to promote early brain injury (EBI) after subarachnoid hemorrhage (SAH). Previous studies have demonstrated that valproic acid (VPA) decreased brain injury in a prechiasmatic injection model of SAH in mice. It should be noted that the beneficial effects of VPA and the underlying mechanisms have not been fully elucidated. To characterize the effects of VPA on BBB disruption and neural apoptosis and to determine mechanisms involved in EBI after SAH. An endovascular perforation model was used to induce SAH in rats. VPA (300 mg/kg) was promptly administered after SAH induction, and the same dose was given 12 hours later. Quercetin (100 mg/kg), an inhibitor of heat shock protein 70 (HSP70), was injected into the peritoneum 2 hours before SAH induction. Mortality, SAH grades, neurological function, Evans Blue extravasation, brain edema, transmission electron microscopy, Western blot, double fluorescence labeling, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling staining also were used. VPA treatment decreased BBB disruption and brain edema, attenuated neural apoptosis, and improved neurobehavioral functions in EBI after SAH. Double fluorescence labeling indicated that matrix metallopeptidase 9 (MMP-9) was located predominately in neurons and endothelial cells. VPA upregulated the expression of HSP70, effectively decreased the expression and activity of MMP-9, and reduced claudin-5 and occludin degradation. Meanwhile, VPA also upregulated the expression of phosphorylated Akt and bcl-2. Both the anti-BBB disruption and antiapoptotic effects of VPA were abolished by quercetin. VPA prevented BBB disruption and alleviated neural apoptosis after SAH. The action of VPA appeared to be mediated though the HSP70/MMPs and HSP70/Akt pathways. BBB, blood-brain barrierEBI, early brain injuryHSP, heat shock proteinMMP, matrix metalloproteinasePBS, phosphate-buffered salineSAH, subarachnoid hemorrhageTUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelingVPA, valproic acid.

MCP-1-induced ERK/GSK-3β/Snail signaling facilitates the epithelial-mesenchymal transition and promotes the migration of MCF-7 human breast carcinoma cells.

Monocyte chemoattractant protein-1 (MCP-1) is a chemotactic cytokine that can bind to its receptor cysteine-cysteine chemokine receptor 2 (CCR2) and plays an important role in breast cancer cell metastasis. However, the molecular mechanisms underlying MCP-1-induced alterations in cellular functions during tumor progression are poorly understood. Here, we showed that MCP-1 stimulated the epithelial-mesenchymal transition (EMT) and induced the tumorigenesis of breast cancer cells by downregulating E-cadherin, upregulating vimentin and fibronectin, activating matrix metallopeptidase-2 (MMP-2), and promoting migration and invasion. Moreover, MCP-1 treatment reduced glycogen synthase kinase-3β (GSK-3β) activity via the MEK/ERK-mediated phosphorylation of serine-9 in MCF-7 cells. The inhibition of MEK/ERK by U0126 attenuated the MCP-1-induced phosphorylation of GSK-3β and decreased the expression of Snail, an EMT-related transcription factor, leading to the inhibition of MCF-7 cell migration and invasion. Inactivation of GSK-3β by LiCl (lithium chloride) treatment notably increased MMP-2 activity, vascular endothelial growth factor expression and EMT of MCF-7 cells. These findings revealed that MCP-1-induced EMT and migration are mediated by the ERK/GSK-3β/Snail pathway, and identified a potential novel target for therapeutic intervention in breast cancer.

Protective effect of simvastatin on impaired intestine tight junction protein ZO-1 in a mouse model of Parkinson's disease.

Recently, several studies showed that gastrointestinal tract may be associated with pathophysiology of Parkinson's disease (PD). Intestine tight junction protein zonula occluden-1 (ZO-1) is an important component of intestinal barrier which can be degraded by matrix metallopeptidase 9 (MMP-9). In our previous study, a significant decline in ZO-1 was observed along with enhanced MMP-9 activity in the duodenum and distal colon of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. In this study, the protective effect of simvastatin on ZO-1 was investigated using an MPTP mouse model of PD. Seven days after the end of MPTP application, the expression level of ZO-1 was evaluated by immunohistochemistry. The protein expression levels of ZO-1 and MMP9 were detected by Western blotting. Meanwhile, MMP-9 activity was analyzed by gelatin zymography. MPTP treatment led to a decrease in the expression of ZO-1, which was accompanied by elevated MMP-9 activity. Treatment with simvastatin could partly reverse the MPTP-induced changes in ZO-1 expression and reduce MMP-9 protein and activity. Taken together, these findings suggest that simvastatin administration may partially reverse the impairment of ZO-1 induced by MPTP via inhibiting the activity of MMP9, fortify the impaired intestinal barrier and limit gut-derived toxins that pass across the intestinal barrier.

The Role of Congestion in Cardiorenal Syndrome Type 2: New Pathophysiological Insights into an Experimental Model of Heart Failure

Background: In cardiorenal syndrome type 2 (CRS2), the role of systemic congestion in heart failure (HF) is still obscure. We studied a model of CRS2 [monocrotaline (MCT)-treated rats] secondary to pulmonary hypertension and right ventricular (RV) failure in order to evaluate the contribution of prevalent congestion to the development of kidney injury. Methods: Ten animals were treated with MCT for 4 weeks until they developed HF. Eleven animals were taken as controls. Signs of hypertrophy and dilatation of the right ventricle demonstrated the occurrence of HF. Brain natriuretic peptide (BNP), serum creatinine (sCreatinine), both kidney and heart neutrophil gelatinase-associated lipocalin (NGAL), matrix metallopeptidase 9 (MMP9), serum cytokines as well as kidney and heart cell death, as assessed by TUNEL, were studied. Results: Rats with HF showed higher BNP levels [chronic HF (CHF) 4.8 ± 0.5 ng/ml; controls 1.5 ± 0.2 ng/ml; p < 0.0001], marked RV hypertrophy and dilatation (RV mass/RV volume: CHF 1.46 ± 0.31, controls 2.41 ± 0.81; p < 0.01) as well as pleural and peritoneal effusions. A significant increase in proinflammatory cytokines and sCreatinine was observed (CHF 3.06 ± 1.3 pg/ml vs. controls 0.54 ± 0.23 pg/ml; p = 0.04). Serum (CHF 562.7 ± 93.34 ng/ml vs. controls 245.3 ± 58.19 ng/ml; p = 0.02) as well as renal and heart tissue NGAL levels [CHF 70,680 ± 4,337 arbitrary units (AU) vs. controls 32,120 ± 4,961 AU; p = 0.001] rose significantly, and they were found to be complexed with MMP9 in CHF rats. A higher number of kidney TUNEL-positive tubular cells was also detected (CHF 114.01 ± 45.93 vs. controls 16.36 ± 11.60 cells/mm²; p = 0.0004). Conclusion: In this model of CHF with prevalent congestion, kidney injury is characterized by tubular damage and systemic inflammation. The upregulated NGAL complexed with MMP9 perpetuates the vicious circle of kidney/heart damage by enhancing the enzymatic activity of MMP9 with extracellular matrix degradation, worsening heart remodeling.

Regulation of metastasis of bladder cancer cells through the WNT signaling pathway.

Bladder cancer (BC) is the most popular malignant urinary cancer, with the highest incidence and mortality of all genitourinary system tumors worldwide. To date, the molecular regulation of the metastasis of BC remains ill defined. Here, we examined the levels of matrix metallopeptidase 9 (MMP9) and nuclear β-catenin in the BC specimen. We used lithium chloride (LiCl) to inhibit cytosol β-catenin phosphorylation and degradation to increase nuclear β-catenin levels in BC cells. We used IWP-2 to enhance cytosol β-catenin phosphorylation and degradation to decrease nuclear β-catenin levels in BC cells. We examined MMP9 levels in these experimental settings by quantitative reverse transcription-PCR (RT-qPCR), Western blot, and ELISA. The cell invasiveness was evaluated by Transwell cell assay. We found significantly higher levels of MMP9 and nuclear β-catenin in human BC specimen with metastasis, compared to those without metastasis. Moreover, a strong correlation was detected between MMP9 and nuclear β-catenin. LiCl significantly increased nuclear β-catenin, resulting in MMP9 activation in BC cells. IWP-2 significantly decreased nuclear β-catenin, resulting in MMP9 inhibition in BC cells. MMP9 regulated cell invasiveness. Together, these data suggest that the WNT signaling pathway regulates metastasis of BC through activation of MMP9. Therapies targeting the WNT signaling pathway may be a promising treatment for BC.

Fibrinogen Regulates MMP‐9 Processing of VEGF‐induced Angiogenesis and Tumor Growth in Osteosarcoma

PurposeOsteosarcoma (OGS) is the most common primary malignant bone tumor in children and adolescents. Traditionally, fibrin, the essential structural matrix protein of a blood clot, is thought to enhance tumor adhesion and provide a matrix to support tumor growth. Evidence strongly supports that major angiogenic factors, such as vascular endothelial growth factor (VEGF) and matrix metallopeptidase 9 (MMP‐9), can bind fibrin in a concentration‐dependent manner. Therefore, we designed experiments to determine the effects of fibrin on OGS tumor angiogenesis by interacting with VEGF and MMP‐9.MethodsAggressive murine OGS cells are intratibially injected into Balb/C mice. OGS establishment is determined by radiographic analysis, micro‐CT, histology, and angiography.ResultsOpposed to traditional beliefs surrounding fibrin's role in tumor establishment, this work has shown that the loss of fibrin promotes the proliferation and vascularity of OGS, therefore promoting tumor establishment and disease progression.imageTherefore, we have confirmed that fibrin does play a role in tumor angiogenesis through its interaction with VEGF, however we have also shown for the first time that MMP‐9 is a novel intermediary factor for this interaction.ConclusionsIn this study, we have demonstrated that fibrin can inhibit tumor angiogenesis by inhibiting MMP‐9 activity and by sequestering VEGF that is cleaved by MMP‐9. This finding demonstrates a novel synergistic relationship between fibrin and MMP‐9 in the progression of OGS, while also identifying new therapeutic targets.

Abstract T P232: Combination Therapy With Taurine Plus Tissue Plasminogen Activator in a Rat Model of Embolic Stroke

Background: Tissue plasminogen activator (tPA) is the only FDA-approved therapy for acute ischemic stroke, but it has narrow therapeutic window (within 4.5h) and increased risks of intracerebral hemorrhage. Taurine, an endogenous 2-aminoethansulfolic amino acid, exhibits a plethora of physiological functions. It has been found to protect against ischemic brain injury in animal models of stroke by diverse mechanisms. Strikingly, published data show that the therapeutic window of taurine was at least 8h after the onset of ischemic stroke, thus potentially allowing for delayed or more effective thrombolysis. In this study we investigated whether combination therapy with taurine plus tPA can block hemorrhagic transformation and thus extend the time window of tPA after delayed tPA treatment for ischemic stroke. Methods and Results: Male adult rats (330-380g) were subjected to embolic middle cerebral artery occlusion and then randomized into different treatment groups: saline injected at 4h; taurine (50 mg/kg) at 4h, tPA (10 mg/kg) at 6h, and combination therapy with taurine at 4h plus tPA at 6h after the onset of ischemia. Delayed 6-hour tPA did not decrease infarction but instead worsened brain hemorrhage. Combining taurine with delayed 6-hour tPA reduced tissue infarction, attenuated blood-brain barrier disruption by reduced degradation of tight junction proteins (claudin-5 and collagen IV), and ameliorated brain hemorrhage. These protective effects are correlated with decreased brain matrix metallopeptidase-9 activity possibly through inactivation of NF-kB and CD147 (a potent inducer of MMPs) in the postischemic brain, particularly in the cerebral microvessels (using structurally intact microvessels isolated from the brain). Conclusion: The present study demonstrates for the first time that combination therapy with taurine may attenuate delayed tPA-associated hemorrhagic transformation and extend tPA treatment time windows in a clinically relevant thromboembolic stroke model. Inhibition of MMP-9 in brain microvessels via inactivation of NF-κB and CD147 signaling may underlie the protective mechanisms of taurine.

The combination of astragalus membranaceus and ligustrazine ameliorates micro-haemorrhage by maintaining blood–brain barrier integrity in cerebrally ischaemic rats

Ethnopharmacological relevanceHaemorrhagic transformation is an asymptomatic event that frequently occurs after following ischaemic stroke, particularly when pharmaceutical thrombolysis is used. However, the mechanism responsible for haemorrhagic transformation remains unknown, and therapeutics have not been identified. In this study, we administered a combination of astragalus membranaceus and ligustrazine to rats with cerebral ischaemia that had undergone thrombolysis. We analysed the effect of this combination on the attenuation of haemorrhagic transformation and the maintenance of blood–brain barrier integrity. MethodsA rat model of focal cerebral ischaemia was induced with autologous blood clot injections. Thrombolysis was performed via the intravenous injection of rt-PA. Astragalus membranaceus, ligustrazine or a combination of Astragalus membranaceus and ligustrazine was administered immediately after the clot injection. The cerebral infarct area, neurological deficits, blood–brain barrier integrity, and cerebral haemorrhage status were determined after 3, 6 and 24h of ischaemia. The ultrastructure of the blood–brain barrier was examined with a transmission electron microscope. The expression of tight junction proteins, including claudin-1, claudin-5, occludin, and zonula occludens-1, and matrix metallopeptidase-9 activation was further evaluated in terms of their roles in the protective effects of the combination drug on the integrity of the blood–brain barrier. ResultsIschaemia-induced Evans blue leakage and cerebral haemorrhage were markedly reduced in the combination drug-treated rats compared to the rats treated with either astragalus membranaceus or ligustrazine alone (p<0.05). The disruption of the ultrastructure of the blood–brain barrier and the neurological deficits were ameliorated by the combination treatment (p<0.05). The reductions in the expression of laudin-1, claudin-5, occludin, and ZO-1 were smaller in the rats that received the combination treatment. In addition, MMP-9 activity was suppressed in the combination-treated rats compared to the controls (p<0.05). ConclusionsTreatment with a combination of astragalus membranaceus and ligustrazine alleviated ischaemia-induced micro-haemorrhage transformation by maintaining the integrity of the blood–brain barrier.

RNA interference-mediated knockdown of RhoGDI2 induces the migration and invasion of human lung cancer A549 cells via activating the PI3K/Akt pathway.

Rho GDP dissociation inhibitor 2 (RhoGDI2) has been identified as a tumor suppressor gene for cellular migration and invasion. However, the underlying mechanism and effector targets of RhoGDI2 in lung cancer are still not fully understood. In this study, a vector-expressed small hairpin RNA (shRNA) of RhoGDI2 was transfected into the human lung cancer cell line A549. After the successful transfection, the down-regulation of RhoGDI2 promoted the proliferation, migration, and invasion of lung cancer cells in vitro through the increasing expression and activities of the matrix metallopeptidase 9 (MMP-9) and PI3K/Akt pathways. Transiently transfecting the small interfering RNA (siRNA) of MMP-9 into the RhoGDI2 shRNA cells reduced the MMP-9 expression. Both transfecting the siRNA and adding the MMP-9 antibody into the RhoGDI2 shRNA cells led to a decrease in the invasion and migration of the lung cancer cells. The blockade of the PI3K/Akt pathway by LY294002 resulted in abolishment of the effects of RhoGDI2 shRNA in Akt phosphorylation and MMP-9 expression. This result suggests that the down-regulated RhoGDI2 contributed to the migration and invasion of the lung cancer cell line via activating the PI3K/Akt pathway and the ensuing increase in the expression and activity of MMP-9. In conclusion, we report that the shRNA-mediated knockdown of RhoGDI2 induces the invasion and migration of lung cancer due to cross-talk with the PI3K/Akt pathway and MMP-9. Verifying the role and molecular mechanism of the participation of RhoGDI2 in the migration and invasion of lung cancer may provide a target for better treatment.

WFDC1 Is a Key Modulator of Inflammatory and Wound Repair Responses

WFDC1/ps20 is a whey acidic protein four-disulfide core member that exhibits diverse growth and immune-associated functions invitro. Invivo functions are unknown, although WFDC1 is lower in reactive stroma. A Wfdc1-null mouse was generated to assess core functions. Wfdc1-null mice exhibited normal developmental and adult phenotypes. However, homeostasis challenges affected inflammatory and repair processes. Wfdc1-null mice infected with influenza A exhibited 2.75-log-fold lower viral titer relative to control mice. Wfdc1-null infected lungs exhibited elevated macrophages and deposition of osteopontin, a potent macrophage chemokine. In wounding studies, Wfdc1-null mice exhibited an elevated rate of skin closure, and this too was associated with elevated deposition of osteopontin and macrophage recruitment. Wfdc1-null fibroblasts exhibited impaired spheroid formation, elevated adhesion to fibronectin, and an increased rate of wound closure invitro. This was reversed by neutralizing antibody to osteopontin. Osteopontin mRNA and cleaved protein was up-regulated in Wfdc1-null cells treated with lipopolysaccharide or polyinosinic-polycytidylic acid coordinate with constitutively active matrix metallopeptidase-9 (MMP-9), a protease that cleaves osteopontin. These data suggest that WFDC1/ps20 modulates core host response mechanisms, in part, via regulation of osteopontin and MMP-9 activity. Release from WFDC1 regulation is likely a key component of inflammatory and repair response mechanisms, and involves the processing of elevated osteopontin by activated MMP-9, and subsequent macrophage recruitment.

Targeting filamin B induces tumor growth and metastasis via enhanced activity of matrix metalloproteinase-9 and secretion of VEGF-A.

Filamins regulate cell locomotion and associate with diverse signaling molecules. We have recently found that targeting filamin A (FLNA) reduces RAS-induced lung adenocarcinomas. In this study, we explored the role of another major filamin isoform, filamin B (FLNB), in tumor development. In contrast to FLNA, we report that targeting FLNB enhances RAS-induced tumor growth and metastasis which is associated with higher matrix metallopeptidase-9 (MMP-9) and extracellular signal-regulated kinase (ERK) activity. Flnb deficiency in mouse embryonic fibroblasts results in increased proteolytic activity of MMP-9 and cell invasion mediated by the RAS/ERK pathway. Similarly, silencing FLNB in multiple human cancer cells increases the proteolytic activity of MMP-9 and tumor cell invasion. Furthermore, we observed that Flnb-deficient RAS-induced tumors display more capillary structures that is correlated with increased vascular endothelial growth factor-A (VEGF-A) secretion. Inhibition of ERK activation blocks phorbol myristate acetate-induced MMP-9 activity and VEGF-A secretion in vitro. In addition, silencing FLNB in human ovarian cancer cells increases secretion of VEGF-A that induces endothelial cells to form more vascular structures in vitro. We conclude that FLNB suppresses tumor growth and metastasis by regulating the activity of MMP-9 and secretion of VEGF-A which is mediated by the RAS/ERK pathway.

MMP9 activation triggered by epidermal growth factor induced FoxO1 nuclear exclusion in non-small cell lung cancer

The molecular mechanism underlying activation of matrix metallopeptidase 9 (MMP9) in non-small cell lung cancer (NSCLC) cells, which controls cancer invasiveness and metastasis, remains elusive. Here, we reported a strong correlation of epidermal growth factor receptor (EGFR) and MMP9 levels in NSCLC patients. Thus, we used a human NSCLC line, A549, to examine whether there is a causal link between EGFR and MMP9 activation. We found that EGF-induced activation of EGFR in A549 cells activated MMP9, resulting in an increase in cancer invasiveness. An EGFR inhibitor efficiently blocked this EGF-induced activation of MMP9 and, consequently, increased cancer invasiveness. Moreover, an inhibitor for phosphatidylinositol 3-kinase (PI3K)/Akt, but not an inhibitor for mitogen-activated protein kinase, or an inhibitor for Jun N-terminal kinase, significantly inhibited the epidermal growth factor (EGF)-induced activation of MMP9, suggesting that PI3K/Akt signaling cascades may be responsible for EGF-activated MMP9. We further dissected the pathway and found that nuclear exclusion of a major Akt downstream target, FoxO1, occurred by EGF-induced Akt activation, which could be inhibited by either EGFR inhibitor or by PI3K/Akt inhibitor. In a loss of function, expression of a constitutive nuclear form of FoxO1 significantly inhibited MMP9 activation induced by EGF. Taken together, these findings suggest that EGF/EGFR signaling activates downstream PI3K/Akt to induce FoxO1 nuclear exclusion, which activates MMP9 to promote NSCLC invasiveness. Thus, Akt and FoxO1, in addition to the well-known EGFR, appear to be promising therapeutic targets for preventing the metastasis of NSCLC.

Role of P2X Purinoceptor 7 in Neurogenic Pulmonary Edema after Subarachnoid Hemorrhage in Rats

IntroductionNeurogenic pulmonary edema (NPE) is an acute and serious complication after subarachnoid hemorrhage (SAH) with high mortality. The present study aimed to test the therapeutic potential of brilliant blue G (BBG), a selective P2X purinoceptor 7 (P2X7R) antagonist, on NPE in a rat SAH model.MethodsSAH was induced by endovascular perforation. 86 Sprague-Dawley rats were randomly divided into sham, vehicle-, or BBG-treatment groups. Mortality, body weight, SAH grading, neurological deficits, NPE clinical symptoms, and pulmonary index were measured at 24 hours following SAH. Western blot, gelatin zymography, lung histopathology, and immunofluorescence staining were performed in the left lung lobe to explore the underlying mechanisms at 24 hours post-surgery.ResultsThe incidence of clinical symptoms was correlated with pulmonary index. P2X7R and the marker of alveolar type I epithelial cells (the mucin-type glycoprotein T1-α) immunoreactivities were generally co-localized. BBG administration decreased mature interleukin-1β, myeloperoxidase, and matrix metallopeptidase-9 activation, but increased tight junction proteins, such as ZO-1 and occludin, which ameliorated pulmonary edema via anti-inflammation and improved neurological deficits.ConclusionP2X7R inhibition prevented NPE after SAH by attenuating inflammation. Thus, BBG is a potential therapeutic application for NPE after SAH and warrants further research.

Brefeldin A Effectively Inhibits Cancer Stem Cell-Like Properties and MMP-9 Activity in Human Colorectal Cancer Colo 205 Cells

Cancer stem cells (CSCs) are a small subset of cancer cells with indefinite potential for self-renewal and the capacity to drive tumorigenesis. Brefeldin A (BFA) is an antibiotic that is known to block protein transport and induce endoplasmic reticulum (ER) stress in eukaryotic cells, but its effects on colorectal CSCs are unknown. We investigated the inhibitory effect of BFA on human colorectal cancer Colo 205 cells. We found that BFA effectively reduced the survival of suspension Colo 205 cells (IC50 = ~15 ng/mL) by inducing apoptosis, and inhibited the clonogenic activity of Colo 205 CSCs in tumorsphere formation assay and soft agar colony formation assay in the same nanogram per milliliter range. We also discovered that at such low concentrations, BFA effectively induced endoplasmic reticulum (ER) stress response as indicated by the increased mRNA expression of ER stress-related genes, such as glucose-regulated protein 78 (GRP78), X-box binding protein 1 (XBP1), and C/EBP homologous protein (CHOP). Finally, we found that BFA reduced the activity of matrix metallopeptidase 9 (MMP-9). These findings suggest that BFA can effectively suppress the progression of colorectal cancer during the tumorigenesis and metastasis stages. These results may lead to the development of novel therapies for the treatment of colorectal cancer.

High expression level of interleukin-6 led to elevated hemorrhagic risk of cerebral arteriovenous malformations

To explore the mechanistic roles of interleukin-6 (IL-6) and matrix metallopeptidase 9 (MMP-9) in cerebral arteriovenous malformation (AVM). A total of 31 AVM patients were admitted into Beijing Tiantan Hospital from October 1, 2010 to October 1, 2011, including 14 ruptured and 17 non-ruptured ones. Tissue samples were obtained from all 31 patients and 30 epileptics undergoing temporal lobectomy. The blood samples were obtained from all 31 patients preoperatively and 30 healthy controls. Western blot was employed to measure transcription factors nuclear factor-κB (NF-κB), IκB and MMP-9 in tissues. And immunofluorescence was performed to measure the tissue expression of MMP-9 in each group. Gelatin zymography was used to detect the tissue expressions of activated MMP-9 and MMP-2. The blood levels of IL-6 in the ruptured group were significantly higher than those in the non-ruptured and control groups (33.2±4.8 vs 23.8±1.2 ng/L, P<0.05; 33.4±4.8 vs 15.6±1.0 ng/L, P<0.01). Protein expression in the non-ruptured group was greater than that in the normal and ruptured groups (1.20±0.35 vs 0.34±0.07; 1.20±0.35 vs 0.31±0.09, unit:molecular weight ratio of MMP-9 and β-actin), and gelatin zymography showed that the activity of MMP-9 was significantly higher in the ruptured than the non-ruptured and control groups (0.98±0.07 vs 0.40±0.09; 0.98±0.07 vs 0.30±0.07, unit: ratio of MMP-9 and standard). In the ruptured group, active MMP-2 expression was significantly higher than that in the other groups. IL-6 may stimulate the transformation of MMP-9 into activated form in ruptured AVM tissues and thus lead to an elevated hemorrhage risk of AVM.

Downregulation of Cyclophilin A by siRNA diminishes non-small cell lung cancer cell growth and metastasis via the regulation of matrix metallopeptidase 9

BackgroundCyclophilin A (CypA) is a cytosolic protein possessing peptidyl-prolyl isomerase activity that was recently reported to be overexpressed in several cancers. Here, we explored the biology and molecular mechanism of CypA in non-small cell lung cancer (NSCLC).MethodsThe expression of CypA in human NSCLC cell lines was detected by real-time reverse transcription PCR. The RNA interference-mediated knockdown of CypA was established in two NSCLC cell lines (95C and A549). 239836 CypA inhibitor was also used to suppress CypA activity. Tumorigenesis was assessed based on cellular proliferation, colony formation assays, and anchorage-independent growth assays; metastasis was assessed based on wound healing and transwell assays.ResultsSuppression of CypA expression inhibited the cell growth and colony formation of A549 and 95C cells. CypA knockdown resulted in the inhibition of cell motility and invasion. Significantly, we show for the first time that CypA increased NSCLC cell invasion by regulating the activity of secreted matrix metallopeptidase 9 (MMP9). Likewise, suppression of CypA with 239836 CypA inhibitor decreased cell proliferation and MMP9 activity.ConclusionsThe suppression of CypA expression was correlated with decreased NSCLC cell tumorigenesis and metastasis.

Iatrogenic extracellular matrix disruption as a local trigger for postoperative ileus

BackgroundActive matrix metallopeptidase 9 (MMP-9) disruption of the extracellular matrix (ECM) plays an important role in inflammatory disorders. In this study, we investigated the inflammatory role of MMP-9 and the ECM breakdown product hyaluronan as a trigger for the postoperative intestinal inflammatory response of postoperative ileus. MethodsWe performed a standardized intestinal surgical manipulation on rats to produce ileus assessed by the oral non-digestible fluorescein isothiocyanate–dextran transit assay. We studied isolated intestinal muscularis extracts for mRNA expressions of interleukin 6 (IL-6), MMP-9 and CD44. We quantified peritoneal MMP-9 activity using zymography, and quantified peritoneal fluid and serum for hyaluronan and tissue inhibitor of metalloproteinase 1 levels by enzyme-linked immunosorbent assay (ELISA). We cultured peritoneal macrophages and exposed them to peritoneal fluid or synthetic hyaluronan for ELISA analysis of IL-6 and macrophage inflammatory protein-1α. ResultsTransit was significantly delayed after surgical manipulation, and extracts of the isolated jejunal and colonic muscularis demonstrated a significant induction of IL-6, MMP-9, and CD44 mRNAs compared with controls. Zymography confirmed significant MMP-9 activity in peritoneal fluid compared with controls. Enzyme-linked immunosorbent assay measurements showed a significant up-regulation in hyaluronan and tissue inhibitor of metalloproteinase 1 in the peritoneal fluid and serum. In addition, ELISA and reverse transcriptase–polymerase chain reaction measurements of peritoneal macrophages stimulated with postsurgical peritoneal fluid and synthetic hyaluronan resulted in higher expressions of IL-6 and macrophage inflammatory protein-1α in the macrophage supernatant. ConclusionsOur results confirm that MMP-9 disruption in the ECM with hyaluronan release and muscularis CD44 receptor induction has the potential to trigger muscularis proinflammatory cascades that cause postoperative ileus. Matrix metallopeptidase 9 inhibition may be a novel therapeutic approach to limit postoperative ileus.

Cleaved high molecular weight kininogen inhibits tube formation of endothelial progenitor cells via suppression of matrix metalloproteinase 2.

Endothelial progenitor cells (EPCs) contribute to postnatal neovascularization, thus promoting wide interest in their therapeutic potential in vascular injury and prevention of their dysfunction in cardiovascular diseases. Cleaved high molecular weight kininogen (HKa), an activation product of the plasma kallikrein-kinin system (KKS), inhibits the functions of differentiated endothelial cells including in vitro and in vivo angiogenesis. In this study, our results provided the first evidence that HKa is able to target EPCs and inhibits their tube forming capacity. We determined the effect of HKa on EPCs using a three-dimensional vasculogenesis assay. Upon stimulation with vascular endothelial growth factor (VEGF) alone, EPCs formed vacuoles and tubes, and differentiated into capillary-like networks. As detected by gelatinolytic activity assay, VEGF stimulated secretion and activation of matrix metallopeptidase 2 (MMP-2), but not MMP-9, in the conditioned medium of 3D culture of EPCs. Specific inhibition or gene ablation of MMP-2, but not MMP-9, blocked the vacuole and tube formation by EPCs. Thus, MMP-2 is selectively required for EPC vasculogenesis. In a concentration-dependent manner, HKa significantly inhibited tube formation by EPCs and the conversion of pro-MMP-2 to MMP-2. Moreover, HKa completely blocked the association between pro-MMP-2 and alphavbeta3 integrin, and its inhibition of MMP-2 activation was dependent on the presence of alphavbeta3 integrin. In a purified system, HKa did not directly inhibit MMP-2 activity. HKa inhibits tube forming capacity of EPCs by suppression of MMP-2 activation, which may constitute a novel link between activation of the KKS and EPC dysfunction.