Inhibition Of MMP-9 Activity Research Articles

Matrix Metalloproteinases are required for membrane motility and lumenogenesis during Drosophila heart development.

Matrix Metalloproteinases (Mmps) degrade glycoproteins and proteoglycans of the extracellular matrix (ECM) or cell surface and are crucial for morphogenesis. Mmps and their inhibitors are expressed during early stages of cardiac development in vertebrates and expression is altered in multiple congenital cardiomyopathies such as cardia bifida. Drosophila genome encodes two copies of Mmps, Mmp1 and Mmp2 whereas in humans up to 25 Mmps have been identified with overlapping functions. We investigated the role of Mmps during embryonic heart development in Drosophila, a process which is morphogenetically similar to early heart tube formation in vertebrates. We demonstrate that the two Mmps in Drosophila have distinct and overlapping roles in cell motility, cell adhesion and cardiac lumenogenesis. We determined that Mmp1 and Mmp2 promote Leading Edge membrane dynamics of cardioblasts during collective migration. Mmp2 is essential for cardiac lumen formation, and mutants generate a cardia bifida phenotype. Mmp1 is required for luminal expansion. Mmp1 and Mmp2 both localise to the basal domains of cardiac cells, however, occupy non-overlapping domains apically. Mmp1 and Mmp2 regulate the proteoglycan composition and size of the apical and basal ECM, yet only Mmp2 is required to restrict ECM assembly to the lumen. Mmp1 negatively regulates the size of the adhesive Cadherin cell surface domain, whereas in a complementary fashion, Mmp2 negatively regulates the size of the Integrin-ECM domain and thereby prescribes the domain to establish and restrict Slit morphogen signalling. Inhibition of Mmp activity through ectopic expression of Tissue Inhibitor of Metalloproteinase in the ectoderm blocks lumen formation. Therefore, Mmp expression and function identifies ECM differentiation and remodelling as a key element for cell polarisation and organogenesis.

Dehydroandrographolide inhibits oral cancer cell migration and invasion through NF-κB-, AP-1-, and SP-1-modulated matrix metalloproteinase-2 inhibition

Background and purposeOral cancer is a type of head and neck cancer that is characterized by cancerous tissue growth in the oral cavity. Andrographolide and dehydroandrographolide (DA) are the two principal components of Andrographis paniculata (Burm.f.) Nees and are the main contributors to its therapeutic properties. However, the pharmacological activities of DA remain unclear. Experimental approachIn this study, we used wound closure assay and Boyden chamber assay to determine the effects of DA on oral cancer cell migration and invasion. Key resultsDA treatment significantly inhibited the migration and invasion abilities of SCC9 cells in vitro. Gelatin zymography and Western blotting results revealed that DA inhibited MMP-2 activity and reduced its protein levels. DA inhibited the phosphorylation of ERK1/2, p38, and JNK 1/2 in SCC9 cells. According to the mRNA levels detected using real-time PCR, DA inhibited MMP-2 expression in SCC9 cells. This inhibitory effect was associated with the upregulation of the TIMP-2 and downregulation of NF-κB, AP-1, and SP-1 expression. In addition, DA suppressed carcinoma-associated epithelial–mesenchymal transition in SCC9 cells. Finally, DA administration effectively suppressed MMP-2 expression and tumor metastases in the oral carcinoma xenograft mouse model in vivo. Conclusions & implicationsDA inhibits the invasion of human oral cancer cells and is a potential chemopreventive agent against oral cancer metastasis.

The natural retinoprotectant chrysophanol attenuated photoreceptor cell apoptosis in an N-methyl-N-nitrosourea-induced mouse model of retinal degenaration

Retinitis pigmentosa (RP) is an inherited photoreceptor-degenerative disease, and neuronal degeneration in RP is exacerbated by glial activation. Cassia seed (Jue-ming-zi) is a traditional herbal medicine commonly used to treat ocular diseases in Asia. In this report, we investigated the retina-protective effect of chrysophanol, an active component of Cassia seed, in an N-methyl-N-nitrosourea (MNU)-induced mouse model of RP. We determined that chrysophanol inhibited the functional and morphological features of MNU-induced retinal degeneration using scotopic electroretinography (ERG), optical coherence tomography (OCT), and immunohistochemistry analysis of R/G opsin and rhodopsin. Furthermore, TUNEL assays revealed that chrysophanol attenuated MNU-induced photoreceptor cell apoptosis and inhibited the expression of the apoptosis-associated proteins PARP, Bax, and caspase-3. In addition, chrysophanol ameliorated reactive gliosis, as demonstrated by a decrease in GFAP immunolabeling, and suppressed the activation of matrix metalloproteinase (MMP)-9-mediated gelatinolysis. In vitro studies indicated that chrysophanol inhibited lipopolysaccharide (LPS)-induced iNOS and COX-2 expression in the BV2 mouse microglia cell line and inhibited MMP-9 activation in primary microglia. Our results demonstrate that chrysophanol provided neuroprotective effects and inhibited glial activation, suggesting that chrysophanol might have therapeutic value for the treatment of human RP and other retinopathies.

Effect of the oral iron chelator deferiprone in diabetic nephropathy rats.

The aim of the present study was to investigate the effects of the iron chelator deferiprone in diabetic nephropathy (DN) rats and the mechanisms involved. Thirty-two male Wistar rats (180-220 g, 6 weeks old) were randomly divided into a control group, a DN group and two DN groups treated with either 50 or 100 mg/kg per day deferiprone. The DN group was established by feeding of a high-carbohydrate-fat diet and injection of 35 mg/kg streptozotocin into the vena caudalis. The duration of deferiprone treatment was 20 weeks. Histopathological changes were detected by hematoxylin-eosin and Masson staining, as well as transmission electron microscopy. Levels of nuclear factor (NF)-κB, monocyte chemotactic protein (MCP)-1, matrix metalloproteinase (MMP)-9, tissue-specific inhibitor of metalloproteinase (TIMP)-1, cyclo-oxygenase (COX)-2, and nitrotyrosine were determined in kidney tissues using reverse transcription-polymerase chain reaction (RT-PCR), western blotting, and immunohistochemistry. Histopathological observations showed that deferiprone treatment alleviated inflammation infiltrates and collagenous fibrosis in DN rats. Results from RT-PCR and western blotting indicated that deferiprone inhibited the expression of NF-κB, MCP-1, COX-2, and nitrotyrosine, which were overexpressed in DN rats. Immunohistochemistry showed that the mechanism of deferiprone action may involve regulation of MMP-9 and TIMP-1. Decreased MMP-9 expression and increased TIMP-1 expression in DN rats were significantly promoted and inhibited by deferiprone, respectively. Iron chelation by oral deferiprone has a renoprotective effect in DN rats by relieving oxidative stress, inflammation, and fibrosis, which is related to the cytokines NF-κB, MCP-1, MMP-9, TIMP-1, COX-2, and nitrotyrosine.

Left Ventricular Unloading After Acute Myocardial Infarction Reduces MMP/JNK Associated Apoptosis and Promotes FAK Cell-Survival Signaling

The mechanism underlying left ventricular remodeling and reverse remodeling in the setting of mechanical support following acute myocardial infarction (MI) is unclear. We tested the hypothesis that left ventricular assist device (LVAD) unloading can decrease apoptotic signals after MI. An MI model was created in 16 sheep by coronary artery ligation. Eight were unloaded with a LVAD during the first 2 weeks after MI and observed for 10 more weeks. Myocardial tissue was collected from the nonischemic adjacent zone and the remote zone. Proteins in theapoptotic matrix metalloproteinases (MMPs)-2/c-Jun N-terminal kinase (JNK) and prosurvival β1D-integrin/focal adhesion kinase (FAK) pathway were quantified. Increased TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) positive nuclei were observed in the MI group and to a lesser extent in the LVAD group (6.18 ± 0.26 versus 0.82 ± 0.18; p< 0.05). Pro-MMP-2, MMP-2, JNK, and phosphorylated (p)-JNK were all elevated in the adjacent zone of the MI-only group but not in the adjacent zone of the LVAD-supported group. There were higher levels of prosurvival p-FAK in the LVAD-supported group than in the MI group. MMP-2/JNK apoptotic and β1D-integrin/FAK survival pathways are activated in the nonischemic adjacent zone after MI in adult sheep. LVAD unloading of approximately 50% cardiac output for 2 weeks attenuates remodeling in part by its negative effect on stretch-induced apoptosis and inhibition of MMP-2 activity.

Aesculetin의 항산화 활성과 MMP-9 활성 억제를 통한 암세포 침윤 억제

Aesculetin의 항산화 활성과 MMP-9 활성 억제를 통한 암세포 침윤 억제

인간 신장암 Caki세포에서 Par-4에 의한 MMP-2 활성 저해를 통한 세포 이동 조절

Par-4는 다양한 세포사멸 자극에 세포 사멸을 조절하고, 종양 억제기능을 가지고 있다. 그러나, Par-4에 의한 암세포의 이동 및 침윤에 대한 연구는 수행되지 않았다. 본 연구에서 Par-4단백질의 과발현이 인간 신장암 Caki세포에서 MMP-2의 활성화를 억제하지만 MMP-9 활성에는 영향을 주지 않았다. Par-4에 의한 MMP-2의 활성 억제는 leucine zipper domain이 결실된 Par-4 에서는 확인되지 않았다. Par-4 siRNA를 이용한 knock-down 실험에서 PMA 처리 시 세포이동 및 침윤 증가함을 확인하였다. Par-4의 과발현과 knock-dwon에서 MMP-2 mRNA 발현의 변화를 확인 할 수 없었다. 이 점은 Par-4 매개의 MMP-2 활성 억제는 전사 후 조절을 통하여 야기됨을 추측 할 수 있다. The prostate-apoptosis-response-gene-4 (Par-4) protein has been identified as an effector of cell death in response to various apoptotic stimuli in prostate cancer cells. We found that overexpression of Par-4 by stable transfection inhibits cell migration and invasion in Caki cells. The expression of various matrix metalloproteinases (MMPs) has been implicated in the invasion and metastasis of cancer cells. In this study, we investigated whether ectopic expression of Par-4 modulates MMP-2 expression and activity in human renal carcinoma Caki cells. We found that overexpression of Par-4 markedly inhibited MMP-2 activity, but not MMP-9 activity. However, loss of the leucine zipper domain of Par-4 (Par-4 &#x394;LZ#1 and #2) did not inhibit MMP-2 activity. Further, knock-down of Par-4 with the corresponding siRNA resulted in increased invasion and metastasis of renal carcinoma Caki cells. Interestingly, overexpression or knock-down of Par-4 did not affect the expression levels of MMP-2 mRNA. Taken together, our findings suggest that Par-4 may inhibit MMP-2 activity through its post-transcriptional regulation in renal carcinoma Caki cells.

Minocycline is More Potent Than Tetracycline and Doxycycline in Inhibiting MMP-9 in Vitro

Background: Tetracyclines are antimicrobial agents that possess anti-inflammatory and matrix metalloproteinase (MMP) inhibitory effects. Tetracycline, doxycycline, and minocycline have shown different potencies in inhibiting various MMPs. MMPs are a conserved family of zinc-dependent endopeptidases, which are involved in various physiologic and pathologic conditions. MMP-9 is among the most important proteases involved in the development of cardiovascular disease, cancer, and inflammatory disease Objectives: Considering the central role of MMP-9 in the above-mentioned diseases and the variable inhibitory potentials of routine tetracyclines, including tetracycline, doxycycline, and minocycline, this study measured the inhibitory effect of these routine tetracycline agents on MMP-9 activity obtained from U-937 cell cultures using the zymography method. Materials and Methods: An MMP-9-rich culture medium of U-937 cells was used as the source of the enzyme. Serial dilutions of 5, 20, 100, 200, and 500 µM of each drug were placed in zymography incubation buffer. Gelatin zymography was run, and densitometry of the bands was performed using Image J software. The resulting data were used to draw dose-response curves using GraphPad Prism software. Results: Our results showed that tetracyclines inhibited MMP-9 activity with IC50 values of 40.0, 10.7, and 608.0 µM for tetracycline, minocycline, and doxycycline, respectively. Minocycline showed the highest potency in MMP-9 inhibition (P < 0.0001), while tetracycline was found to be more potent than doxycycline in MMP-9 inhibition based on the zymography experiments (P < 0.0001). Conclusions: Our results suggest that tetracyclines dose-dependently inhibit MMP-9 activity, and among the common clinical tetracyclines, minocycline provides the most potent inhibition of excess MMP-9 activity and its consequences.

Alkaloid rich fraction from Nelumbo nucifera targets VSMC proliferation and migration to suppress restenosis in balloon-injured rat carotid artery

AimsRestenosis- an adverse consequence following angioplasty, and atherosclerosis are characterized by abnormal vascular smooth muscle cell (VSMC) proliferation and migration leading to neo-intima formation. In the present study, we investigated the inhibitory effects of alkaloid rich fraction (ARF) from Nelumbo nucifera and isolated compound neferine on platelet-derived growth factor (PDGF-BB) induced VSMC proliferation and migration in vitro and neo-intima formation in a rat carotid artery injury model. MethodsPDGF-BB induced VSMC proliferation and migration was assessed using colorimetric assay and modified Boyden chamber method respectively. Gene expression of cell cycle associated molecules was determined by reverse transcription-polymerase chain reaction (RT-PCR). The signaling molecules such as PDGF-Rβ, extracellular regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK), P38, metalloproteinase (MMP)-9 and nuclear factor-kappa B (NF-κB) were determined by western blot analysis. Stress fiber formation was evaluated using immunofluorescence microscopy. The rat carotid artery balloon injury model was performed to assess the effect of ARF on neo-intima formation. ResultsARF possessed the strongest anti-oxidant activities. The anti-proliferative activity of both ARF and neferine was due to suppression of cyclin D1, cyclin E and cyclin-dependent kinase (Cdk) gene expression. Moreover, ARF and neferine inhibited PDGF-Rβ, ERK1/2, JNK and P38 activations and NF-κB translocation. Also, ARF and neferine inhibited VSMC migration by inhibiting MMP-9 activity without affecting cytoskeleton remodeling. In a rat carotid artery injury model, ARF inhibited neo-intima formation. ConclusionOur results indicate that ARF targets VSMC proliferation and migration to attenuate neo-intima formation by inhibition of PDGF-Rβ mediated signaling.

A leading role for NADPH oxidase in an in-vitro study of experimental autoimmune encephalomyelitis.

Myelin oligodendrocyte glycoprotein peptide fragment 35-55 (MOG35-55) is a major autoantigen inducing experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis that is characterized by blood-brain barrier (BBB) disruption. Various experimental approaches have employed MOG35-55 in vivo; however, in vitro BBB models using MOG35-55 are rarely reported. We investigated MOG35-55 exposure effects with complete Freund's adjuvant (CFA) and pertussis toxin (PTX) on brain endothelial cells and elucidated the relationships among NADPH oxidase, MMP-9, ICAM-1, and VCAM-1. These 4 factors significantly increased in MOG35-55+CFA+PTX-exposed endothelial cells compared with the control cells. NADPH oxidase inhibition using apocynin reduced MMP-9 activity, ICAM-1, and VCAM-1. MMP-9 inhibitor I decreased expression of ICAM-1 and VCAM-1, and both anti-ICAM-1 and anti-VCAM-1 inhibited MMP-9 activity. Inhibitions of MMP-9, ICAM-1, and VCAM-1 did not change NADPH oxidase activity. Although inhibition of these 4 factors decreased BBB permeability in cells, inhibition of NADPH oxidase exhibited the highest decrease among these. NADPH oxidase directly influenced MMP-9, ICAM-1, and VCAM-1, but not vice versa. MMP-9 and the cell adhesion molecules reversibly affected each other. In conclusion, NADPH oxidase-derived superoxide elevated expression of MMP-9, ICAM-1, and VCAM-1, and these interactions can finally result in increases of BBB permeability in MOG35-55+CFA+PTX-exposed endothelial cells.

Anti-Tumor Effect of Pinus massoniana Bark Proanthocyanidins on Ovarian Cancer through Induction of Cell Apoptosis and Inhibition of Cell Migration.

Pinus massoniana bark proanthocyanidins (PMBPs), an active component isolated from Pinus massoniana bark, has been reported to possess a wide range of biochemical properties. Here, we investigated the anti-tumor effect of PMBPs on ovarian cancer. The results indicated that PMBPs significantly reduced the growth of ovarian cancer cells and induced dose-dependent apoptosis. The underlying mechanisms involved were elucidated to include the loss of mitochondrial membrane potential, down-regulation of the anti-apoptotic protein Bcl-2 and the activation of Caspase 3/9, suggesting that PMBPs triggered apoptosis through activation of mitochondria-associated apoptotic pathway. In addition, wound healing and transwell chamber assays revealed that PMBPs could suppress migration and invasion of ovarian cancer cells. PMBPs dramatically inhibited MMP-9 activity and expression, blocked the activity of NFκB and the activation of ERK1/2 and p38 MAPK. Our findings suggest that PMBPs has the potential to be developed as an anti-tumor drug for ovarian cancer treatment and/ or disease management.

Polyphenol mixtures of Euphorbia supina the inhibit invasion and metastasis of highly metastatic breast cancer MDA-MB-231 cells.

The Korean prostrate spurge Euphorbia supina is abundant in polyphenols and has been used as a folk medicine in Korea against a variety of diseases. Thus, we aimed to investigate the effect of polyphenol mixtures of Korean Euphorbia supina (PES) on the invasion and metastasis of highly metastatic breast cancer MDA-MB-231 cells. Firstly, PES showed no cytotoxicity on cancer cells and endothelial cells (ECs) at the doses of 0.1-10 µg/ml, but showed significant cytotoxicity from 50 µg/ml. Thus, we performed subsequent experiments with PES at doses up to 5 µg/ml. PES dose‑dependently suppressed epithelial-mesenchymal transition by downregulating the mesenchymal markers, Snail1 and N-cadherin, showing significant inhibition from 1 and 5 µg/ml, respectively. In addition, PES significantly inhibited MMP-9 activity and LOX release induced by TNF-α at 5 µg/ml. Then, we determined the effect of PES on the expression of adhesion molecules and VE-cadherin phosphorylation. The results showed that PES effectively reduced TNF-α-mediated VCAM-1 expression but not ICAM expression both in the MDA-MB-231 cells and ECs, resulting in the reduced adhesion of MDA-MB-231 to ECs. Finally, PES effectively inhibited MDA-MB-231 cell invasion through ECs, suggesting that PES may serve as a therapeutic agent against cancer metastasis with minimal cytotoxicity to normal cells.

The Anti-Inflammatory Effects of the Methanolic Extract and Fractions from Davilla elliptica St. Hil. (Dilleniaceae) on Bothrops jararaca Envenomation.

Inflammation and haemorrhage are the main characteristics of tissue injury in botropic envenomation. Although some studies have shown that anti-venom prevents systemic reactions, it is not efficient in preventing tissue injury at the site of the bite. Therefore, this work was undertaken to investigate the anti-inflammatory effects of the methanolic extract and fractions from D. elliptica and to evaluate the role of matrix metalloproteinases (MMPs) in this process. Effects of the extract and fractions from D. elliptica were evaluated using a carrageenan-induced paw oedema model in rats, and leukocyte rolling was visualized by intravital. The quantification of MMPs activities (MMP-2 and MMP-9) extracted from the dermis of mice treated with extract and fractions alone or incubated with venom was determined by zymographic analyses. Our results show that intraperitoneal (i.p.) injection of fractions significantly reduced paw oedema after the carrageenan challenge. Treatment with the tannins fraction also resulted in considerable inhibition of the rolling of leukocytes and this fraction was able to decrease the activation of MMP-9. These results confirmed the anti-inflammatory activity of the methanolic extract and tannins fraction of D. elliptica and showed that the dermonecrosis properties of B. jararaca venom might be mediated through the inhibition of MMP-9 activity.

Green tea catechin inhibits the activity and neutrophil release of Matrix Metalloproteinase-9

Green tea (Camellia sinensis; 綠茶 lǜ chá) extracts have been shown to possess anti-oxidant and anti-inflammatory effects in various cell types. Green tea extract (GTX) has been shown to significantly inhibit the activity of collagenase-3 (matrix metalloproteinase-13 (MMP-13)) in vitro. MMPs, such as MMP-9, are known to be involved in many inflammatory diseases including periodontal disease. GTX and a major catechin, epigallocatechin-gallate (EGCG), were examined for their ability to inhibit purified MMP-9 activity and its release from stimulated neutrophils.Methanol extract of Green tea and commercially purchased EGCG (>95 % purity) were tested in vitro for their ability to inhibit MMP-9 activity and/or its release from neutrophils using a β-casein cleavage assay and gelatin zymography, respectively. Statistical analysis was performed by Student's t-test.GTX and EGCG at 0.1% (w/v) completely inhibited the activity of MMP-9. In addition, GTX and EGCG (0.1 %) significantly inhibited (p < 0.001) the release of MMP-9 from formyl-Met-Leu-Phe (FMLP)-stimulated human neutrophils by 62.01% ± 6.717 and 79.63% ± 1.308, respectively. The inhibitory effects of GTX and EGCG occurred in unstimulated neutrophils (52.42% ± 3.443 and 62.33% ± 5.809, respectively). When the inhibitory effect of EGCG was further characterized, it significantly inhibited the release of MMP-9 from the FMLP-stimulated human neutrophils in a dose-dependent manner.The effects of GTX and EGCG on MMPs could be extrapolated to clinical/in vivo studies for the development of oral care products to prevent or treat chronic inflammatory diseases including periodontal diseases.

Synthesis, crystal structure and effect of indeno[1,2-b]indole derivatives on prostate cancer in vitro. Potential effect against MMP-9

A highly regiospecific synthesis of a series of indenoindoles is reported, together with X-ray studies and their activity against human prostate cancer cells PC-3 and LNCaP in vitro. The most effective compound 7,7-dimethyl-5-[(3,4-dichlorophenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9bhexahydro-indeno[1,2-b]indole-9,10-dione 7q reduced the viability in both cell lines in a time and dose-dependent manner. Inhibitory effects were also observed on the adhesion, migration, and invasion of the prostate cancer cells as well as on clonogenic possibly by inhibition of MMP-9 activity. Molecular docking of 7q and 6k into MMP-9 human active site was also performed to determine the probable binding mode.

Delivery of lipid micelles into infarcted myocardium using a lipid-linked matrix metalloproteinase targeting peptide.

There is a great need for delivery strategies capable of efficiently localizing drugs to the damaged myocardium that do not require direct intramyocardial injection of therapeutic molecules. In the work discussed here, we exploited the myocardium-specific upregulation of matrix metalloproteinases (MMPs) that occurs during myocardium remodeling by designing a micellar vehicle containing an MMP-targeting peptide (MMP-TP). The binding of MMP-TP to MMP was evaluated with purified MMP-2 protein and U-937 cells induced to overexpress MMP. Inhibition of MMP-2 activity was not observed in the presence of unmodified micelles but was pronounced at a 5 mol % MMP-TP ligand density. In a FACS analysis, MMP-TP micelles containing 5 mol % of the MMP-targeting peptide showed ∼10-fold higher binding to activated U937 cells than plain micelles and micelles containing a control peptide with two amino acid replacements. MMP-TP-micelles and plain micelles were injected intravenously into C57BL/6 mice 1, 3, and 7 days after the induction of a myocardial infarction (MI). Immunohistochemistry performed on heart tissue sections revealed that MMP-TP-micelles colocalize with both MMP and infiltrating macrophages. MMP-TP micelles showed significantly enhanced accumulation to the necrotic area of the heart after MI on days 3 and 7 when compared to plain micelles and negative control peptide micelles. This is coincident with the measured temporal profile of MMP gene expression in the heart after MI. These results suggest that MMP-TP micelles are candidates for the development of targeted regenerative heart therapeutics because of their ability to target the infarcted myocardium in a MMP dependent manner.

TIMP-1 inhibition of occludin degradation in Caco-2 intestinal cells: a potential protective role in necrotizing enterocolitis.

Necrotizing enterocolitis (NEC), a common intestinal disease affecting premature infants, is a major cause of morbidity and mortality. Previous reports indicate an upregulation of intestinal matrix metalloproteinases (MMPs) activity that may play key roles on the higher permeability of the intestinal barrier, typical to NEC. Recently, TIMP-1, a natural inhibitor of MMP's, was found to be over expressed in preterm human breast milk (HBM). Previous studies have shown that infants fed with HBM have a significant reduction in the incidence of NEC. The aim of the present study was to investigate the possible role that TIMP-1 may play on the maintenance of tight junctions and therefore the gut barrier integrity. Timp-1-treated Caco-2 intestinal cells were tested for MMP-2 enzymatic activity and cell junction integrity. TIMP-1 inhibited MMP-2 activity, which induced a significant increase in the expression of occludin but not of claudin-4. TIMP-1 did not affect apoptosis. One of the putative mechanisms associated with HBM protection against NEC is mediated by TIMP-1, which downregulates MMP-2 activity, inhibits the degradation of occluding, and preserves tight junctions and gut barrier integrity.

Abstract W P238: Selective Deletion of Pi3-kinase Gamma in the Brain Inhibits Tissue Plasminogen Activator-induced Brain Hemorrhage

Background: PI3-kinase gamma (PI3Kγ), a key modulator in inflammation, is involved in many inflammatory diseases. We recently reported that PI3Kγ deficiency protects the brain from ischemic injury in PI3Kγ-/- mice by reducing blood-brain barrier damage and tissue infarction through inhibition of NF-kB-dependent inflammation, mainly involved in reduced MMP-9 activity in ischemic brain endothelium, microglia activation, and neutrophil infiltration into the ischemic brain. Meanwhile, we reported that ischemic brain endothelium, activated microglia, and infiltrating neutrophils represent the major cellular sources of PI3Kγ expressed in the brain within 24 h after cerebral ischemia. Brain hemorrhage is a serious complication of recombinant tissue plasminogen activator (tPA) therapy for ischemic stroke. In this study, we further investigated potential involvement of PI3Kγ, in particular the expression of PI3Kγ in the brain, in tPA-induced brain hemorrhage after ischemic stroke. Methods and Results: Focal ischemic stroke was induced by transient middle cerebral artery occlusion and reperfusion. PI3Kγ deficiency in PI3Kγ-/- mice not only reduced tissue infarction but also almost completely blocked brain hemorrhage induced by delayed tPA treatment at 6 hours after stroke. Bone marrow chimeras reveal that chimeric mice lacking PI3Kγ in the brain (WT BM →KO) blocked the delayed tPA-induced brain hemorrhage effectively as found in PI3Kγ-/- mice while chimeric mice lacking PI3Kγ expressed in leukocytes (KO BM →WT) had minimal effect on the tPA-induced brain hemorrhage. These effects could be attributed to a profound inhibition of MMP-9 activity in ischemic brain endothelium found in both PI3Kγ-/- mice and (WT BM →KO) chimeric mice but not in (KO BM →WT) chimeric mice, as determined by double IHC staining of MMP-9 in brain endothelial cells and by zymographic analysis of MMP-9 activity using microvessels isolated from the brain. Conclusion: Our findings suggest that PI3Kγ in the brain may play a predominant role in delayed tPA-induced brain hemorrhage after ischemic stroke. Our future study will define the role of PI3Kγ derived from brain endothelial cells versus microglia by generation of tissue/cell-specific knockout mice using the Cre-loxP system.

Low-dose multi target suppression of cardiac ischemia/reperfusion pathology for improved physiological outcome

More recent evidence in my lab shows that, in addition to the pathological role of MMP-2 in I/R, MMP-2 can also regulate MLC levels under physiological conditions. I have evidence showing that robust inhibition of MMP-2 activity increases MLC levels (impairing MLC turnover) resulting in increased contractility above homeostatic levels (normal). This emphasizes the importance of only targeting pathological activity while maintaining physiological homeostasis. I have also shown that I/R increases phosphorylation and nitration/nitrosylation of MLC proteins leading to increased degradation by MMP-2. These MLC modifications play important physiological roles in addition to targeting the protein for degradation. Nitration of MLC has been reported in the process of vascular aging, and the phosphorylation of MLC2 controls the sensitivity of myofilaments to calcium in the regulation of heart contractility. Thus, maintaining thephysiological activity of MMP-2 and functional MLC modifications while simultaneously providing protection from I/R is a difficult set of tasks. I suggest, that in addition to the existing approaches in clinical practice for protection and treatment of heart injury from MI or I/R; simultaneous pharmacological suppression of MMP-2 activity and MLC nitration/nitrosylation/phosphorylation will provide improved protection of the heart from ischemic injury while limiting drug associated side effects. My studies have shown that a mixture of low concentrations of potential therapeutic agents (inhibitors of MMP-2 activity and MLC nitration/nitrosylation/ phosphorylation) act in a synergistic manner to provide protection from reperfusion injury. Thus, development of a “drug cocktail” or a “super pill” to protect the hearts of human patients from I/R injury may be a legitimate approach. Additional drugs that target different pathways should also be explored for their potential to act synergistically for ultimate protection. Drugs such as inhibitors of apoptosis, c-Jun N-terminal kinases (JNK), glycogen synthase kinase (GSK) or modulators of energy metabolism may also prove beneficial. Because the basic molecular and cellular mechanisms of all I/R injury and protection are essentially similar throughout the body, this concept is important to all pathologies involving I/R; including transplantation or reattachment of limbs.

MiR-146a targets Fos expression in human cardiac cells.

ABSTRACTmiR-146a is a microRNA whose transcript levels are induced in the heart upon activation of NF-κB, a transcription factor induced by pro-inflammatory molecules (such as TNF-α) that is strongly related to the pathogenesis of cardiac disorders. The main goal of this study consisted of studying new roles of miR-146a in cardiac pathological processes caused by the pro-inflammatory cytokine TNF-α. Our results demonstrate that miR-146a transcript levels were sharply increased in cardiac ventricular tissue of transgenic mice with specific overexpression of TNF-α in the heart, and also in a cardiomyocyte cell line of human origin (AC16) exposed to TNF-α. Among all the in silico predicted miR-146a target genes, Fos mRNA and protein levels notably decreased after TNF-α treatment or miR-146a overexpression. These changes correlated with a diminution in the DNA-binding activity of AP-1, the Fos-containing transcription factor complex. Interestingly, AP-1 inhibition was accompanied by a reduction in matrix metalloproteinase (MMP)-9 mRNA levels in human cardiac cells. The specific regulation of this MMP by miR-146a was further confirmed at the secretion and enzymatic activity levels, as well as after anti-miR-mediated miR-146a inhibition. The results reported here demonstrate that Fos is a direct target of miR-146a activity and that downregulation of the Fos–AP-1 pathway by miR-146a has the capacity to inhibit MMP-9 activity. Given that MMP-9 is an AP-1 target gene involved in cardiac remodeling, myocardial dysfunction and progression of heart failure, these findings suggest that miR-146a might be a new and promising therapeutic tool for treating cardiac disorders associated with enhanced inflammation in the heart.