Levels Of Matrix Metalloproteinase-9 Activity Research Articles

Elucidating the role of MMP9 in hyperhomocysteinemia induced degradation of the neurovascular unit

AbstractBackgroundVascular contributions to cognitive impairment and dementia (VCID) is one of the leading causes of dementia. Hyperhomocysteinemia has been identified as a risk factor for VCID though the mechanism behind this connection has yet to be determined. Hyperhomocysteinemia increases inflammation inducing the upregulation of matrix metalloproteinase 9 (MMP9) activity. MMP9 has been shown to degrade several substrates along the neurovascular unit including claudin 5, collagen 4, occludin and β‐dystroglycan. I hypothesize that increased MMP9 activity following the induction of hyperhomocysteinemia leads to the cleavage of important anchoring proteins along the neurovascular unit weakening the integrity of the blood brain barrier which contributes to the progression of VCID pathology.MethodC57BL6 WT and MMP9 knockout mice were placed on a control diet or a diet deficient in vitamins B6, B12 and folate and enriched in methionine to induce hyperhomocysteinemia for 4, 8, 12 and 16 weeks. Western blot analysis was performed to investigate the substrates of MMP9 including claudin, occludin, and β‐dystroglycan.ResultStudies are underway to examine if MMP9 activation contributes to blood brain barrier dysfunction directly by degrading tight junction proteins leading to a weakened endothelial cell barrier and/or indirectly by cleaving proteins responsible for maintaining the connection between astrocytic end feet and the endothelial basement membrane.ConclusionOur findings suggest that increased levels of MMP9 activity may impact several aspects of the neurovascular unit to contribute to VCID pathology. Further investigations focused on inhibition of MMP9 activation could provide the field with an important target for mitigating progression of this disease.

Increased matrix metalloproteinase 9 activity correlates with flow-mediated intraluminal thrombus deposition and wall degeneration in human abdominal aortic aneurysm

ObjectiveWe have previously demonstrated that human abdominal aortic aneurysm (AAA) rupture occurs in zones of low wall shear stress where flow recirculation and intraluminal thrombus (ILT) deposition are increased. Matrix metalloproteinase-9 (MMP-9) is involved in the pathogenesis of AAA via its lytic effect on collagen and elastin. We hypothesize that flow-mediated ILT deposition promotes increased local inflammatory and MMP-9 activity that leads to AAA wall degeneration. The purpose of this study was to examine the correlation between predicted pulsatile flow dynamics and regional differences in MMP-9, elastin, collagen, and ILT deposition in human AAA. MethodsFull-thickness aortic tissue samples were collected from 24 patients undergoing open AAA repair. Control infrarenal aortic tissue was obtained from 6 patients undergoing aortobifemoral bypass. Full-thickness aortic tissue and ILT were assessed for MMP-9 levels using a cytokine array assay. Histologic and immunohistochemical assessment of inflammation, collagen and elastin content, and MMP-9 levels were also measured. Three-dimensional AAA geometry was generated from computed tomography angiogram (CTA) images using Mimics software and computational fluid dynamics was used to predict pulsatile aortic blood flow. ResultsThe majority of AAA showed eccentric ILT deposition which was correlated with predicted recirculation blood flow (R2 = –0.17; P < .05). The regions of high ILT were associated with significant increases in inflammation and loss of elastin and collagen compared with regions of low ILT, or with control tissue. MMP-9 was significantly higher in areas of high ILT deposition compared with areas devoid of ILT. Tissue MMP-9 was correlated with the thickness of ILT deposition (R2 = 0.46; P < .05), and was also present in high levels in thick compared with thin ILT. ConclusionsWe have shown a correlation between flow-mediated ILT deposition with increased tissue levels of MMP-9 activity, increased inflammatory infiltrate, and decreased elastin and collagen content in stereotactically sampled human AAA, suggesting that ILT deposition is associated with local increases in proteolytic activity that may preferentially weaken and promote rupture at selected regions.

High MMP‐9 activity levels in fragile X syndrome are lowered by minocycline

Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by lack of the FMR1 protein, FMRP, a translational repressor. Its absence leads to up-regulation of locally translated proteins involved in synaptic transmission and plasticity, including the matrix metalloproteinase-9 (MMP-9). In the Fmr1 knock-out (KO), a mouse model of FXS, an abnormal elevated expression of MMP-9 in the brain was pharmacologically down-regulated after treatment with the tetracycline derivative minocycline. Moreover, the rescue of immature dendritic spine morphology and a significant improvement of abnormal behavior were associated with down-regulation of MMP-9. Here, we report on high plasma activity of MMP-9 in individuals with FXS. In addition, we investigate MMP-9 changes in patients with FXS who have gone through a minocycline controlled clinical trial and correlate MMP-9 activity to clinical observations. The results of this study suggest that, in humans, activity levels of MMP-9 are lowered by minocycline and that, in some cases, changes in MMP-9 activity are positively associated with improvement based on clinical measures.

TIMP-1 deficiency leads to lethal partial hepatic ischemia and reperfusion injury

Hepatic ischemia and reperfusion injury (IRI) remains an important challenge in clinical orthotopic liver transplantation (OLT). Tissue inhibitor of metalloproteinase-1 (TIMP-1) is the major endogenous regulator of matrix metalloproteinase-9 (MMP-9). In this study we investigated the functional significance of TIMP-1 expression in a well-established mouse model of partial liver IRI. Compared to wildtype mice, TIMP-1(-/-) mice showed further impaired liver function and histological preservation after IRI. Notably, TIMP-1 deficiency led to lethal liver IRI, as over 60% of the TIMP-1(-/-) mice died postreperfusion, whereas all TIMP-1(+/+) mice recovered and survived surgery. Lack of TIMP-1 expression was accompanied by markedly high levels of MMP-9 activity, which facilitates leukocyte transmigration across vascular barriers in hepatic IRI. Indeed, TIMP-1(-/-) livers were characterized by massive leukocyte infiltration and by up-regulation of proinflammatory mediators, including tumor necrosis factor alpha, interferon-gamma, and inducible nitric oxide synthase post-IRI. The inability of TIMP-1(-/-) mice to express TIMP-1 increased the levels of active caspase-3 and depressed the expression of Bcl-2 and the phosphorylation of Akt, emphasizing an important role for TIMP-1 expression on hepatocyte survival. Using independent parameters of regeneration, 5-bromodeoxyuridine incorporation, proliferating cell nuclear antigen expression, and histone H3 phosphorylation, we provide evidence that hepatocyte progression into S phase and mitosis was impaired in TIMP-1-deficient livers after IRI. Inhibition of the cell cycle progression by TIMP-1 deficiency was linked to depressed levels of cyclins-D1 and -E and to a disrupted c-Met signaling pathway, as evidenced by reduced phosphorylated c-Met expression and elevated c-Met ectodomain shedding postliver IRI. These results support a critical protective function for TIMP-1 expression on promoting survival and proliferation of liver cells and on regulating leukocyte recruitment and activation in liver IRI.

Effects of N-acetylcysteine on matrix metalloproteinase-9 secretion and cell migration of human corneal epithelial cells

Matrix metalloproteinase-9 (MMP-9) secreted by corneal epithelial cells has a role in the remodelling of extracellular matrix and migration of epithelial cells. Elevated levels of MMP-9 activity in the ocular surface may be involved in the pathogenesis of corneal diseases. N-acetylcysteine (NAC) has been used to treat corneal diseases, including recurrent epithelial erosions. In this study, its effects on the MMP-9 secretion and human corneal epithelial (HCE) cell migration were evaluated in vitro. Confluent HCE cell cultures were treated with 0-20 mM NAC, and tested for MMP-9 secretion and epithelial cell migration by gelatin zymography and scratch wound assay, respectively. Comparisons between different treatment groups were made using analysis of variance, followed by multiple pairwise comparisons. Twenty mM NAC inhibited the secretion of MMP-9 significantly. Cell migration, assessed after 24 h of wounding, showed a highly significant dose-dependent inhibitory effect. This study shows that NAC reduces MMP-9 production by HCE cells and inhibits cell migration in vitro. This information helps to elucidate the mechanisms by which NAC may be beneficial therapeutically and suggests that NAC may be useful for managing corneal erosions and related conditions.

Macrophage-Mediated Degradation of β-Amyloid via an Apolipoprotein E Isoform-Dependent Mechanism

Recent studies suggest that bone marrow-derived macrophages can effectively reduce beta-amyloid (Abeta) deposition in brain. To further elucidate the mechanisms by which macrophages degrade Abeta, we cultured murine macrophages on top of Abeta plaque-bearing brain sections from transgenic mice expressing PDAPP [human amyloid precursor protein (APP) with the APP(717V>F) mutation driven by the platelet-derived growth factor promoter]. Using this ex vivo assay, we found that macrophages from wild-type mice very efficiently degrade both soluble and insoluble Abeta in a time-dependent manner and markedly eliminate thioflavine-S positive amyloid deposits. Because macrophages express and secrete apolipoprotein E (apoE), we compared the efficiency of Abeta degradation by macrophages prepared from apoE-deficient mice or mice expressing human apoE2, apoE3, or apoE4. Macrophages expressing apoE2 were more efficient at degrading Abeta than apoE3-expressing, apoE4-expressing, or apoE-deficient macrophages. Moreover, macrophage-induced degradation of Abeta was effectively blocked by an anti-apoE antibody and receptor-associated protein, an antagonist of the low-density lipoprotein (LDL) receptor family, suggesting involvement of LDL receptors. Measurement of matrix metalloproteinase-9 (MMP-9) activity in the media from human apoE-expressing macrophages cocultured with Abeta-containing brain sections revealed greater levels of MMP-9 activity in apoE2-expressing than in either apoE3- or apoE4-expressing macrophages. Differences in MMP-9 activity appear to contribute to the isoform-specific differences in Abeta degradation by macrophages. These apoE isoform-dependent effects of macrophages on Abeta degradation suggest a novel "peripheral" mechanism for Abeta clearance from brain that may also, in part, explain the isoform-dependent effects of apoE in determining the genetic risk for Alzheimer's disease.

Cardiac Uptake of Minocycline and Mechanisms for In Vivo Cardioprotection

The ability of minocycline to be transported into cardiac cells, concentrate in normal and ischemic myocardium, and act as a cardioprotector in vivo was examined. We also determined minocycline's capacity to act as a reducer of myocardial oxidative stress and matrix metalloproteinase (MMP) activity. The identification of compounds with the potential to reduce myocardial ischemic injury is of great interest. Tetracyclines are antibiotics with pleiotropic cytoprotective properties that accumulate in normal and diseased tissues. Minocycline is highly lipophilic and has shown promise as a possible cardioprotector. However, minocycline's potential as an in vivo cardioprotector as well as the means by which this action is attained are not well understood. Rats were subjected to 45 min of ischemia and 48 h of reperfusion. Animals were treated 48 h before and 48 h after thoracotomy with either vehicle or 50 mg/kg/day minocycline. Tissue samples were used for biochemical assays and cultured cardiac cells for minocycline uptake experiments. Minocycline significantly reduced infarct size (approximately 33%), tissue MMP-9 activity, and oxidative stress. Minocycline was concentrated approximately 24-fold in normal (0.5 mmol/l) and approximately 50-fold in ischemic regions (1.1 mmol/l) versus blood. Neonatal rat cardiac fibroblasts, myocytes, and adult fibroblasts demonstrated a time- and temperature-dependent uptake of minocycline to levels that approximate those of normal myocardium. Given the high intracellular levels observed and results from the assessment of in vitro antioxidant and MMP inhibitor capacities, it is likely that minocycline acts to limit myocardial ischemic injury via mass action effects.

M. tuberculosis induction of matrix metalloproteinase-9: the role of mannose and receptor-mediated mechanisms.

Mycobacterium tuberculosis (Mtb) infection induces the expression of matrix metalloproteinase-9 (MMP-9) in mouse lungs. In cultured human monocytic cells, Mtb bacilli and the cell wall glycolipid lipoarabinomannan (LAM) stimulate high levels of MMP-9 activity. Here, we explore the cellular mechanisms involved in the induction of MMP-9 by Mtb. We show that infection of THP-1 cells with Mtb caused a fivefold increase in MMP-9 mRNA that was associated with increased MMP-9 activity. MMP-9 induction was dependent on microtubule polymerization and protein kinase activation and was associated with increased DNA binding by the transcription factor activator protein-1 (AP-1), which appeared to be important for MMP-9 expression. We then explored the surface molecules potentially involved in Mtb induction of MMP-9, focusing on ligands of the mannose and beta-glucan receptors. MMP-9 activity was induced by the mannose receptor ligands mannan, zymosan, and LAM, whereas the beta-glucan receptor ligand laminarin was not effective. The most active inducers of MMP-9 activity were the particulate ligand zymosan and LAM. Pretreatment of cells with an anti-mannose receptor monoclonal antibody, but not anti-complement receptor 3, decreased the induction of MMP-9 activity by Mtb bacilli. Together, these results suggest that MMP-9 induction by Mtb occurs by receptor-mediated signaling mechanisms involving the binding of mannosylated ligands to mannose receptors, the modulation by cytoskeletal elements such as microtubules, the activation of protein kinases, and transcriptional activation by AP-1.

High expression of 92 kDa type IV collagenase (matrix metalloproteinase-9) in canine mammary adenocarcinoma

The 92 kDa type VI collagenase (matrix metalloproteinase-9 (MMP-9)) activities on zymography assay were found to be 1–6 times higher in benign tumor breast tissues of 12 canines and 4–26 times higher in adenocarcinoma breast tissues of nine canines than that of control tissues, respectively. A full-length canine MMP-9 cDNA was cloned from the adenocarcinoma tissue by reverse transcription-PCR and 5′- and 3′-RACE. The isolated cDNA contained an open reading frame coding for a polypeptide of 704 amino acids. The predicted protein sequence displayed extensive similarity to that of known MMP-9s and contained a putative signal sequence, a propeptide, an active site with three zinc-binding histidine residues, a calcium-binding domain, a hemopexin region, and three key cysteine residues. Western blotting using MMP-9-specific antibodies prepared against the peptide corresponding to Arg 642–Asp 704 of canine MMP-9 and Northern blotting using a MMP-9-specific cDNA fragment as a probe confirmed that MMP-9 (the 92 kDa protein band) was highly expressed in canine mammary adenocarcinoma tissues. Higher levels of MMP-9 activity were found in the sera of canines with mammary adenocarcinoma. The results indicated that MMP-9 plays an important role in the progression of a canine mammary tumor and that assay of serum MMP-9 is helpful for early diagnosis as progress of adenocarcinoma.

HLA DRB1*1501 and intrathecal inflammation in multiple sclerosis

CD4 T cells are considered to be pivotal in the pathogenesis of multiple sclerosis (MS), and the human leukocyte antigen (HLA) haplotype associated with DRB1*1501 confers susceptibility to MS in patients of Northern European descent. Some previous studies have suggested an association of DRB1*1501 with T- and B-cell reactivity to specific myelin protein peptides, other studies suggested an association with enhanced cytokine production or intrathecal immunoglobulin (Ig) synthesis. In order to further assess the role of DRB1*1501 in the pathogenesis of MS, we studied intrathecal inflammation and T-cell phenotypes in patients with possible onset symptoms or clinically definite MS. Presence of DRB1*1501 was associated with higher levels of cerebrospinal fluid (CSF) inflammation as assessed by IgG synthesis levels and higher levels of matrix metalloproteinase-9 activity. DRB1*1501-positive patients also had a lower percentage of T cells in CSF expressing HLA-DR without co-expressing CD25. These findings suggest that enhanced intrathecal inflammation and an altered T-cell activation status may be of importance in conferring the DRB1*1501-associated susceptibility to MS.

Gelatinase activity in keloids and hypertrophic scars.

Keloids and hypertrophic scars are characterized by excessive deposition of collagen, which may result from insufficient protein degradation. Little is known about the levels of two gelatinases, matrix metalloproteinase-2 (72 kD type IV collagenase) and matrix metalloproteinase-9 (matrix metalloproteinase-9; 92 kD type IV collagenase) in these abnormal scars. The purpose of this study was to determine levels of these proteinases in tissue from hypertrophic scars, keloids, and donor skin. Ten hypertrophic scar samples, 9 keloid samples, and 10 donor skin samples were frozen, pulverized, homogenized, clarified by centrifugation, and analyzed for matrix metalloproteinases by quantitative zymography. Identity of matrix metalloproteinases was determined using a conditioned media reference standard, molecular weight ladders, and Western blotting. Levels of matrix metalloproteinase-9 activity were very low or undetectable in all samples. However, matrix metalloproteinase-2 activity was significantly elevated in keloids and hypertrophic scars vs. donor samples: 2.6 and 3.9-fold increases for latent matrix metalloproteinase-2, 7.8 and 6.9-fold increases for active matrix metalloproteinase-2, respectively. We conclude that little matrix metalloproteinase-9 activity (the gelatinase involved in early tissue repair) is present in keloids and hypertrophic scars, while matrix metalloproteinase-2 activity (the gelatinase involved in prolonged tissue remodeling) is present in donor skin and is significantly increased in hypertrophic scars and keloids.