Elevated Levels Of Matrix Metalloproteinases Research Articles

Interactions between human osteoblasts and prostate cancer cells in a novel 3D in vitro model

Cell-cell and cell-matrix interactions play a major role in tumor morphogenesis and cancer metastasis. Therefore, it is crucial to create a model with a biomimetic microenvironment that allows such interactions to fully represent the pathophysiology of a disease for an in vitro study. This is achievable by using three-dimensional (3D) models instead of conventional two-dimensional (2D) cultures with the aid of tissue engineering technology. We are now able to better address the complex intercellular interactions underlying prostate cancer (CaP) bone metastasis through such models. In this study, we assessed the interaction of CaP cells and human osteoblasts (hOBs) within a tissue engineered bone (TEB) construct. Consistent with other in vivo studies, our findings show that intercellular and CaP cell-bone matrix interactions lead to elevated levels of matrix metalloproteinases, steroidogenic enzymes and the CaP biomarker, prostate specific antigen (PSA); all associated with CaP metastasis. Hence, it highlights the physiological relevance of this model. We believe that this model will provide new insights for understanding of the previously poorly understood molecular mechanisms of bone metastasis, which will foster further translational studies, and ultimately offer a potential tool for drug screening.

Abstract 529: Matrix metalloproteinase expression and secretion in papillary thyroid carcinoma

Abstract In the last decade, the incidence of thyroid cancer has risen ∼84% and mortality ∼34%. The ACS estimates ∼ 37,200 new cases of thyroid cancer, with 1,630 deaths in 2009. Papillary thyroid carcinoma (PTC) is the most common thyroid and endocrine malignancy, accounting for ∼80% of all thyroid cancer. It typically occurs from a gain-of-function mutation in the RET, RAS or BRAF genes which make up a linear signaling cascade for ERK activation. Recurrent or persistent disease prevails in up to 40% of thyroidectomy cases with a poor prognosis when aggressive cancer is evident. Thus, further research into factors contributing to aggressive disease is merited. Matrix metalloproteinases (MMPs) represent a family of secreted proteases with diverse functions implicated in the pathogeneses of aggressive tumors. Their functions include degrading the extracellular matrix (ECM) to facilitate cell migration, cell signaling through ECM cleavage products that can affect apoptosis and cell growth, and they can act directly on growth factor precursors and receptors. MMP upregulation has been demonstrated in many types of cancers yet its pattern of expression is poorly understood in PTC. Analysis of MMPs and tissue inhibitors of metalloproteinases (TIMPs) in the human PTC cell line, BCPAP, and the normal thyroid cell line, NTHY-ori by protein array and real-time RT-PCR revealed significantly elevated levels of MMPs and decreased levels of TIMP-4. Real time RT-PCR demonstrated the following fold increases in expression in BCPAP compared to NTHY-ori: 16. 9 (SE=0.79) for MMP-1, 1.80 (SE=0.41) for MMP-3, 1.40 (SE=0.08) for MMP-9, 7.12 (SE=2.14) for MMP-10, 12.60 (SE=6.90) for MMP-13 and increased levels of TIMP-4 in NTHY-ori compared to BCPAP of 2.38 (SE=1.04). Protein array data from serum free conditioned media of the cell lines reflected a similar pattern of secreted MMP and TIM4 levels. Fold increases in the optical concentration (determined by spot densitometry) of secreted proteins were detected in BCPAP compared to NTHY-ori of 29.48 (SE=1.92) for MMP-1, 16.80 (SE=1.07) for MMP-3, 1.66 (SE=0.07) for MMP-9, 3.66 (SE=0.55) for MMP-10, 1.59 (SE=0.11) for MMP-13 and increased levels of TIMP-4 in NTHY-ori compared to BCPAP of 1.27 (SE=0.06). For the first time, these results demonstrate increased production and secretion of a group of MMPs and down regulation of TIMP-4 which have been characterized in tumor progression in a PTC cell line. Further investigation into the role these agents play in the tumorgenesis of aggressive PTC may yield insight into future treatment and screening alternatives. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 529.

Effect of composition and different fractions of hay dust suspension on inflammation in lungs of heaves-affected horses: MMP-9 and MMP-2 as indicators of tissue destruction

Airway matrix metalloproteinases (MMPs) increase following inhalation of organic dust. The relative contribution of dust components to this elevation is unknown. To identify components of organic dust responsible for elevated MMP levels in equine airways. Bronchoalveolar lavage (BALF) from 7 heaves-susceptible horses, collected 6 h following inhalation challenges with saline, 2 different hay dust suspensions (HDS-1 and -2) and soluble and particulate fractions of HDS-1, were analysed for MMP-2 and -9 using SDS-page gelatin zymography. HDS-1 challenge increased BALF proMMP-9 and total MMP-9. HDS-1 fractions, or the particulate fraction with added lipopolysaccharide, increased BALF proMMP-9 and total MMP-9 in combination, but not when inhaled separately. HDS-2 inhalation elevated BALF complex forms, proMMP-9, active MMP-9, total MMP-9 and total MMP-2. The results suggest synergistic action of soluble and particulate organic dust components. The fact that HDS-1 and HDS-2 had different glucan concentrations supports a role for moulds in the activation of MMP-9. Activation and release of MMPs in response to inhaled moulds are involved in the aetiopathogenesis of heaves. Endotoxin contributes to the synergistic action of the dust components, but the overall MMP response to organic dust inhalation in heaves-susceptible horses largely reflects the mould content of the dust. In the future, inhibition of MMP production and release may offer therapeutic means for treatment and prevention of heaves and recommendations for acceptable dust levels can be given.

Matrix metalloproteinase-dependent shedding of intercellular adhesion molecule-5 occurs with long-term potentiation

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that can be released or activated in a neuronal activity dependent manner. Although pathologically elevated levels of MMPs may be synaptotoxic, physiologically appropriate levels of MMPs may instead enhance synaptic transmission. MMP inhibitors can block long term potentiation (LTP), and at least one family member can affect an increase in the volume of dendritic spines. While the mechanism by which MMPs affect these changes is not completely understood, one possibility is that the cleavage of specific synaptic cell adhesion molecules plays a role. In the present study, we have examined the ability of neuronal activity to stimulate rapid MMP dependent shedding of the intercellular adhesion molecule-5 (ICAM-5), a synaptic adhesion molecule that is thought to inhibit the maturation and enlargement of dendritic spines. Since such cleavage would likely occur within minutes if it were relevant to a process such as LTP, we focused on post stimulus time points of 30 min or less. We show that NMDA can stimulate rapid shedding of ICAM-5 from cortical neurons in dissociated cell cultures and that such shedding is diminished by pretreatment of cultures with inhibitors that target MMP-3 and -9, proteases thought to influence synaptic plasticity. Additional studies suggest that MMP mediated cleavage of ICAM-5 occurs at amino acid 780, so that the major portion of the ectodomain is released. Since reductions in ICAM-5 have been linked to changes in dendritic spine morphology that are associated with LTP, we also examined the possibility that MMP dependent ICAM-5 shedding occurs following high frequency tetanic stimulation of murine hippocampal slices. Results show that the shedding of ICAM-5 occurs in association with LTP, and that both LTP and the associated ICAM-5 shedding are reduced when slices are pretreated with an MMP inhibitor. Together, these findings suggest that neuronal activity is linked to the shedding of a molecule that may inhibit dendritic spine enlargement and that MMPs can affect this change. While further studies will be necessary to determine the extent to which cleavage of ICAM-5 in particular contributes to MMP dependent LTP, our data support an emerging body of literature suggesting that MMPs are critical mediators of synaptic plasticity.

The Cytoplasmic Tail Dileucine Motif LL572 Determines the Glycosylation Pattern of Membrane-type 1 Matrix Metalloproteinase

Membrane-type 1 matrix metalloproteinase (MT1-MMP; MMP-14) drives fundamental physiological and pathological processes, due to its ability to process a broad spectrum of substrates. Because subtle changes in its activity can produce profound physiological effects, MT1-MMP is tightly regulated. Currently, many aspects of this regulation remain to be elucidated. It has recently been discovered that O-linked glycosylation defines the substrate spectrum of MT1-MMP. We hypothesized that a mutual interdependency exists between MT1-MMP trafficking and glycosylation. Lectin precipitation, metabolic labeling, enzymatic deglycosylation, and site-directed mutagenesis studies demonstrate that the LL(572) motif in the cytoplasmic tail of MT1-MMP influences the composition of the complex O-linked carbohydrates attached to the hinge region of the protein. This influence appears to be independent from major effects on cell surface trafficking. MT1-MMP undergoes extensive processing after its synthesis. The origins and the molecular characters of its multiple forms are incompletely understood. Here, we develop and present a model for the sequential, post-translational processing of MT1-MMP that defines stages in the post-synthetic pathway pursued by the protein.

075 Effect of Polyhydrated Ionogen (PHI) on Viability and Matrix Metalloproteinase Levels in Medium of Cultured Cells

It has been postulated that elevated levels of matrix metalloproteinases (MMPs) and reactive oxygen species detected in fluids and biopsies of chronic wounds contribute to the failure of wound to heal by degrading essential growth factors, receptors and extracellular matrix proteins. This stimulated the development of a formulation containing metal ions (polyhydrated ionogen or PHI) and citric acid that reduced reactive oxygen species in cultures of polymorphonuclear leukocytes (J Wound Care 12:10, 2003). The effect of PHI on synthesis of MMPs was studied in cultures of tumor cells and human dermal fibroblasts. Dose response curves of PHI ranging from 0.02% to 10%(W/V) established no toxicity in cultures of human dermal fibroblasts after 24 hours of incubation with PHI at concentrations below 1.25%, as measured by MTT assay. Levels of MMP-2 activity measured by antibody capture assay were reduced approximately 80% in culture medium of Mor-86 glioma tumor cells after 24 hours of exposure to 1% PHI. These results suggest that PHI reduced the synthesis of MMP-2 protein, presumably by reducing transcription and/or translation of MMP-2 gene. Clinical case reports of chronic wounds treated by PHI dressing (DerMax) suggest that PHI treatment stimulated healing and reduced MMP-2 immunostaining in biopsies of the chronic wounds. Acknowledgment: This research was supported by Greystone Medical Group.

Chronic LPS inhalation causes emphysema-like changes in mouse lung that are associated with apoptosis.

Lipopolysaccharide (LPS) is ubiquitous in the environment. Recent epidemiologic data suggest that occupational exposure to inhaled LPS can contribute to the progression of chronic obstructive pulmonary disease. To address the hypothesis that inhaled LPS can cause emphysema-like changes in mouse pulmonary parenchyma, we exposed C57BL/6 mice to aerosolized LPS daily for 4 weeks. By 3 days after the end of the 4-week exposure, LPS-exposed mice developed enlarged airspaces that persisted in the 4-week recovered mice. These architectural alterations in the lung are associated with enhanced type I, III, and IV procollagen mRNA as well as elevated levels of matrix metalloproteinase (MMP)-9 mRNA, all of which have been previously associated with human emphysema. Interestingly, MMP-9-deficient mice were not protected from the development of LPS-induced emphysema. However, we demonstrate that LPS-induced airspace enlargement was associated with apoptosis within the lung parenchyma, as shown by prominent TUNEL staining and elevated cleaved caspase 3 immunoreactivity. Antineutrophil antiserum-treated mice were partially protected from the lung destruction caused by chronic inhalation of LPS. Taken together, these findings demonstrate that inhaled LPS can cause neutrophil-dependent emphysematous changes in lung architecture that are associated with apoptosis and that these changes may be occurring through mechanisms different than those induced by cigarette smoke.

Plasma MMP1, MMP8 and MMP13 expression in breast cancer: protective role of MMP8 against lymph node metastasis

Elevated levels of matrix metalloproteinases (MMPs) have been found to associate with poor prognosis in various carcinomas. This study aimed at evaluating plasma levels of the collagenases MMP1, MMP8 and MMP13 as diagnostic and prognostic markers of breast cancer. Using ELISA, plasma levels of MMP1, MMP8 and MMP13 were measured in 42 control individuals and in 208 patients – of which 21 were inflammatory breast cancer patients – and were correlated with standard clinicopathological data. Plasma MMP1 levels were higher in breast cancer patients than in control individuals, while the opposite was true for MMP8. Plasma MMP13 levels could not be detected. We found a negative correlation of plasma MMP1 with tumour size (P = 0.07); and a positive association of MMP8 with the premenopausal status (P = 0.06), Nottingham Prognostic Index (P = 0.06) and Her2 expression (P = 0.07). Further, a twofold decrease in MMP1 (P = 0.025) and MMP8 (P = 0.007) levels was observed in inflammatory breast cancer patients, a very rare and not well understood aggressive disease. Most interestingly, we observed a peculiar relation between plasma MMP8 levels and lymph node metastasis. We found that both control individuals and patients without lymph node involvement (pN0) have lower plasma MMP8 levels than patients with moderate lymph node involvement (pN1, pN2) (P = 0.001); and that they show a trend for higher MMP8 levels as compared with patients with extensive lymph node metastasis (pN3) and a strong predisposition to distant metastasis. In summary, we observed differences in MMP1 and MMP8 plasma levels between distinct breast cancer patient groups. As it is not clear to date whether MMPs in blood and body fluids have a physiological function per se, we hypothesize that altered levels in blood reflect local changes in the extracellular microenvironment. As such, a positive association of blood MMP levels with clinical characteristics and tumour features reflects a negative association with tissue MMP levels and vice versa. Therefore, our results suggest that both MMP1 and MMP8 in the tumour may contribute to the aggressive phenotype of inflammatory breast carcinomas. Interestingly, our results suggest that tumour MMP8 expression may affect the metastatic behaviour of breast cancer cells with a greater protective effect against lymph node metastasis than against distant metastasis.

Synergistic Collagenase Expression and Cartilage Collagenolysis Are Phosphatidylinositol 3-Kinase/Akt Signaling-dependent

The phosphatidylinositol 3-kinase (PI3K) signaling pathway has emerged as a major regulator of cellular functions and has been implicated in several pathologies involving remodeling of extracellular matrix (ECM). The end stage of inflammatory joint diseases is characterized by excessive ECM catabolism, and in this study we assess the role of PI3K signaling in the induction of collagenolytic matrix metalloproteinases (MMPs) in human chondrocytes. We used the most potent cytokine stimulus reported to promote cartilage ECM catabolism, namely interleukin-1 (IL-1) in combination with oncostatin M (OSM). Both OSM and IL-6 (in the presence of its soluble receptor), but not IL-1 nor leukemia inhibitory factor, induced Akt phosphorylation in human chondrocytes. Inhibition of PI3K signaling using LY294002 blocked IL-1+OSM-mediated Akt phosphorylation, induction of MMP-1 and MMP-13, and cartilage collagenolysis. To further explore the role of downstream substrates within the PI3K pathway, complementary use of small molecule inhibitors and specific small interfering RNAs demonstrated that the PI3K subunit p110alpha and Akt1 were required for MMP-1 mRNA induction. MMP-13 induction was also reduced by loss of function of these molecules and by a lack of p110delta, 3-phosphoinositide-dependent kinase-1 or Akt3. We therefore propose that the activities of specific elements of the PI3K signaling pathway, including Akt, are necessary for the synergistic induction of MMP-1 and MMP-13 and the cartilage breakdown stimulated by IL-1+OSM. Our data provide new insight into the mechanism of synergy between IL-1 and OSM and highlight new therapeutic targets for inflammatory joint diseases that aim to repress the expression of collagenases.

Plasma MMP1 and MMP8 expression in breast cancer: Protective role of MMP8 against lymph node metastasis

BackgroundElevated levels of matrix metalloproteinases have been found to associate with poor prognosis in various carcinomas. This study aimed at evaluating plasma levels of MMP1, MMP8 and MMP13 as diagnostic and prognostic markers of breast cancer.MethodsA total of 208 breast cancer patients, of which 21 with inflammatory breast cancer, and 42 healthy controls were included. Plasma MMP1, MMP8 and MMP13 levels were measured using ELISA and correlated with clinicopathological characteristics.ResultsMedian plasma MMP1 levels were higher in controls than in breast cancer patients (3.45 vs. 2.01 ng/ml), while no difference was found for MMP8 (10.74 vs. 10.49 ng/ml). ROC analysis for MMP1 revealed an AUC of 0.67, sensitivity of 80% and specificity of 24% at a cut-off value of 4.24 ng/ml. Plasma MMP13 expression could not be detected. No correlation was found between MMP1 and MMP8 levels. We found a trend of lower MMP1 levels with increasing tumour size (p = 0.07); and higher MMP8 levels with premenopausal status (p = 0.06) and NPI (p = 0.04). The median plasma MMP1 (p = 0.02) and MMP8 (p = 0.007) levels in the non-inflammatory breast cancer patients were almost twice as high as those found in the inflammatory breast cancer patients. Intriguingly, plasma MMP8 levels were positively associated with lymph node involvement but showed a negative correlation with the risk of distant metastasis. Both controls and lymph node negative patients (pN0) had lower MMP8 levels than patients with moderate lymph node involvement (pN1, pN2) (p = 0.001); and showed a trend for higher MMP8 levels compared to patients with extensive lymph node involvement (pN3) and a strong predisposition to distant metastasis (p = 0.11). Based on the hypothesis that blood and tissue protein levels are in reverse association, these results suggest that MMP8 in the tumour may have a protective effect against lymph node metastasis.ConclusionIn summary, we observed differences in MMP1 and MMP8 plasma levels between healthy controls and breast cancer patients as well as between breast cancer patients. Interestingly, our results suggest that MMP8 may affect the metastatic behaviour of breast cancer cells through protection against lymph node metastasis, underlining the importance of anti-target identification in drug development.

Increased Angiogenesis at the Site of Abdominal Aortic Aneurysm Rupture

Abdominal aortic aneurysm (AAA) rupture is associated with elevated levels of matrix metalloproteinase (MMP). Medial neovascularization is a known characteristic of established AAAs and involves proteolytic degradation of extracellular matrix by MMPs to facilitate endothelial cell proliferation and migration. This study evaluated the extent of neovascularization in abdominal aortic aneurysm rupture. Results indicated upregulation of proangiogenic cytokines and increased medial neovascularization at the aneurysm rupture edge compared with paired aneurysm anterior sac. Further investigations into the role of angiogenesis in aneurysm rupture may open novel therapeutic avenues to prevent aneurysm rupture.

The glycosylation characteristic of hepatoma-associated antigen HAb18G/CD147 in human hepatoma cells

HAb18G/CD147 is a highly glycosylated transmembrane protein belonging to the immunoglobulin superfamily. Our previous studies have demonstrated that overexpressing HAb18G/CD147 enhances the metastatic potentials of human hepatoma cells. In the present study, to investigate the glycosylation characteristic of HAb18G/CD147 in human hepatoma cells, HAb18G/CD147 was first purified from human FHCC-98 hepatoma cells by immunoaffinity chromatography, and then introduced into human fibroblasts culture system for matrix metalloproteinases induction. As a result, the elevated levels of matrix metalloproteinases secreted by fibroblasts were detected by gelatin zymography. The lysates of human hepatoma FHCC-98 cell revealed two major forms of HAb18G/CD147 (43–66 and 35 kDa) by western blot assay. To elucidate whether the variation of molecule size were caused by different glycosylation, two different approaches were employed to accomplish this goal: deglycosylation with N-glycosylation inhibitor tunicamycin or endoglycosidases. A single deglycosylated core protein with molecular weight ∼27 kDa was obtained from both methods. Furthermore, the results of endoglycosidases treatment also showed that two forms of HAb18G/CD147 contain different types of oligosaccharide chains, thus sensitive to different endoglycosidase. In conclusion, the present study demonstrated that purified native HAb18G/CD147 has the bioactivity of stimulating human fibroblasts to produce elevated levels of matrix metalloproteinases, and that the two different forms of HAb18G/CD147 are derived from the single core protein but differ in their degree and types of glycosylation.

Increased plasma MMP9 in integrin α1‐null mice enhances lung metastasis of colon carcinoma cells

Inhibitors of matrix metalloproteinases (MMPs) were developed as anticancer agents based on the observation that MMPs facilitate local tumor spread and metastasis by promoting matrix degradation and cell migration. Unfortunately, these inhibitors were unsuccessful in the clinical treatment of several cancers, including lung cancer. A possible reason contributing to their failure is that MMP activity is critical for the generation of inhibitors of tumor angiogenesis, including angiostatin. Thus, MMPs might play opposing roles in tumor vascularization and invasion. To determine which effect of elevated MMP levels dominates in the progression of metastatic cancer, experimental lung metastasis assays were performed in integrin alpha1-null mice, a genetic model for increased plasma levels of MMP9 and MMP9-generated angiostatin (Pozzi et al., Proc. Natl. Acad. Sci. USA 2000;97:2202-7). We show that while the number of lung colonies in integrin alpha1-null mice was significantly increased compared to their wild-type counterparts, tumor volume was markedly reduced. In vivo treatment with the MMP inhibitor doxycycline resulted in a significant decrease in the number of lung colonies in both genotypes, but the tumors that formed were bigger and more vascularized. Increased tumor vascularization paralleled decreased plasma levels of MMP9 and consequent decreased angiostatin synthesis. These results demonstrate that while inhibition of MMPs prevents and/or reduces tumor invasion and lung metastasis, it has the paradoxical effect of increasing the size and vascularization of metastatic tumors due to decreased generation of inhibitors of endothelial cell proliferation. The continued growth of these large well-vascularized tumors may explain the poor efficacy of MMP inhibitors in lung cancer clinical trials.

LGI1, a Putative Tumor Metastasis Suppressor Gene, Controls in Vitro Invasiveness and Expression of Matrix Metalloproteinases in Glioma Cells through the ERK1/2 Pathway

Gliomas take a number of different genetic routes in the progression to glioblastoma multiforme, a highly invasive variant that is mostly unresponsive to current therapies. Gliomas express elevated levels of matrix metalloproteinases (MMPs), which have been implicated in the control of proliferation and invasion as well as neovascularization. Progressive loss of LGI1 expression has been associated with the development of high grade gliomas. We have shown previously that the forced re-expression of LGI1 in different glioma cells inhibits proliferation, invasiveness, and anchorage-independent growth in cells null for its expression. Here, using Affymetrix gene chip analysis, we show that reexpression of LGI1 in T98G cells results in the down-regulation of several MMP genes, in particular MMP1 and MMP3. LGI1 expression also results in the inhibition of ERK1/2 phosphorylation but not p38 phosphorylation. Inhibition of the MAPK pathway using the pharmacological inhibitors PD98059, U0126, and SB203580 in T98G LGI1-null cells inhibits MMP1 and MMP3 production in an ERK1/2-dependent manner. Treatment of LGI1-expressing cells with phorbol myristate acetate prevents the inhibition of MMP1/3 and restores invasiveness and ERK1/2 phosphorylation, suggesting that LGI1 acts through the ERK/MAPK pathway. Furthermore, LGI1 expression promotes phosphorylation of AKT, which leads to phosphorylation of Raf1(Ser-259), an event shown previously to negatively regulate ERK1/2 signaling. These data suggest that LGI1 plays a major role in suppressing the production of MMP1/3 through the phosphatidylinositol 3-kinase/ERK pathway. Loss of LGI1 expression, therefore, may be an important event in the progression of gliomas that leads to a more invasive phenotype in these cells.

006 Development of a Novel Matrix Metalloproteinase‐Inhibiting Wound Dressing

Rimon Therapeutics develops synthetic therapeutic polymers (Theramers™) that combine bioactivity with a broad range of desirable mechanical and physical properties. The MI Theramer™ is a polymer that modulates the activity of matrix metalloproteinases (MMPs).. Excessive proteolytic cleavage of extracellular matrix and growth factors resulting from elevated levels of MMPs is believed to contribute to the impaired wound healing associated with chronic, non‐healing wounds. MI Theramer™ beads suspended in ThermaGel™(a thermoreversible gel) make up MI‐Gel™ Dressing. The dressing may be applied as a liquid that is poured into a wound, where it will gel on contact. The dressing can be reliquified and removed easily without re‐injuring the wound by cooling it. In vitro inhibition of collagenase type IV (equivalent to MMP‐2) from clostridium histolyticum (Molecular Probes) was measured. A dose‐dependent inhibition of MMP‐2 activity was measured for MI Theramer™ beads alone and suspended in ThermaGel™ indicating that MMP‐2 is able to diffuse into the gel and bind to the beads suspended therein. In vivo efficacy was demonstrated by observing the degradation of glutaraldehyde cross‐linked gelatin tubes using a mouse subcutaneous pouch model. Gelatin zymography on extracts collected from the gelatin tubes co‐implanted with MI Theramer™ beads also showed reduced MMP‐2 and MMP‐9 activity in comparison to controls.The insoluble MI Theramer™ beads were able to modulate MMP activity both in vitro and in vivo. The beads retained their inhibitory activity when they were suspended in a novel thermoreversible gel (ThermaGel™). The combined MI‐Gel™ Dressing may have promise as a novel chronic wound healing product.

Elevated matrix metalloproteinase-2 and -3 production from human diabetic dermal fibroblasts.

Diabetic foot ulcers are characterized by elevated levels of matrix metalloproteinases (MMP), which could lead to excessive matrix breakdown and disruption to healing. It is unknown if this elevation is a function of wound healing, or if it is present within normal skin and a primary contributor to the increased risk of impaired healing. To determine whether diabetic fibroblasts from unwounded skin show elevated MMP production compared with their nondiabetic counterparts. Circular skin biopsies (4 mm diameter) were taken from the inside upper arm of four controls without diabetes and from four subjects with insulin-treated diabetes. Fibroblasts were incubated for a further 72 h and conditioned medium was collected and stored at -20 degrees C. The conditioned medium was assessed by gelatin zymography and Western blotting for MMP-2 and MMP-3. Diabetic dermal fibroblasts showed significantly elevated production of MMP-2 (P < 0.05) and pro-MMP-3 (P < 0.05) when compared with their nondiabetic counterparts. Dermal fibroblasts from normal unwounded skin are characterized by increased MMP production and this may be a primary contributing factor to the increased risk of nonhealing foot ulceration in diabetes.

Elevated levels of matrix metalloproteinase 9 and tissue inhibitor of metalloproteinase 1 during the acute phase of Kawasaki disease.

Kawasaki disease (KD) is an acute, self-limiting, multisystem vasculitis of unknown etiology affecting infants and young children. Unless treated promptly with high-dose intravenous gamma globulin and aspirin, patients frequently develop coronary aneurysms. Previously, matrix metalloproteinase 9 (MMP-9), which is secreted complexed to tissue inhibitor of metalloproteinase 1 (TIMP-1), has been implicated in abdominal aortic aneurysm formation. Since the clinical and pathological features of KD include inflammation and weakening of blood vessels, we analyzed acute- and convalescent-phase paired plasma or serum samples from 31 KD patients, 7 patients who did not completely meet the criteria for KD, and 26 non-KD controls (9 febrile and 17 afebrile patients) for pro-MMP-9 (92 kDa) enzyme activity by gelatin zymography and for active MMP-9 (83 kDa), pro-MMP-9, and TIMP-1 protein levels by enzyme-linked immunosorbent assay. Statistical analysis was performed by using Student t tests, linear regression, and the Wilcoxon rank-sum test. Markedly elevated pro-MMP-9 enzymatic activity, pro-MMP-9 protein levels, and TIMP-1 protein levels were found during the acute phase of illness in patients with clinically established KD and in patients who were suspected of having KD but did not meet all of the criteria. There was no significant difference in active MMP-9 levels. Furthermore, pro-MMP-9 and TIMP-1 protein levels were significantly elevated among KD patients, compared to those of febrile and afebrile non-KD controls. The significantly elevated pro-MMP-9 enzyme and protein levels during the acute phase of KD may reflect vascular remodeling or an inflammatory response to a microbial agent, suggesting a pathophysiological role for MMP-9 in coronary aneurysm formation.

Basement membrane integrity and keratinization in healthy and ulcerated bovine hoof tissue.

Damage to, or deterioration of, the keratinized horn tissue of the bovine hoof claw culminates ultimately in the development of solear ulceration. We have observed abnormal keratin distribution at the site of solear ulceration in the bovine claw that may be due to alteration of the positional cues of the keratinocytes. In this study we have characterized key cell biological changes associated with ulceration in the claw that may precipitate abnormal keratinization. Loss of basement membrane at sites of ulceration was found by immunofluorescent detection of laminin and integrins. In other tissues, basement membrane breakdown results from degradation by matrix metalloproteinases (MMPs). Similarly, elevated levels of MMPs 2 and 9 were observed in ulcerated bovine claw tissue both by zymography and, quantitatively, by assay of enzyme activity. In the sole of claws that contained an ulcer, tissue distal to the ulcer site also had elevated MMP 2 when compared with healthy sole tissue from the same animals, as did sole tissue of claws recovering from ulceration. Tissue inhibitor of metalloproteinase 2 (TIMP 2) was detected by ELISA in healthy tissue. TIMP 2 tended to be lower in diseased tissue distal to ulcer sites, and was significantly lower in ulcerated tissue. MMP 2 was located by immunofluorescence in the dermal and basal epidermal region of sole tissue, in the region of the basement membrane. Increased punctate staining of material in the dermis was associated with ulcerated material. ELISA of TIMP 2 in tissue extracts enriched for dermis or epidermis confirmed that the inhibitor was located predominantly in the dermis. To investigate a possible causal relationship between basement membrane anchorage and epidermal keratinization, the effect of function-blocking antibodies to laminins and integrins was tested in tissue explant cultures prepared from healthy sole tissue. Anti-integrin antibody treatment had no effect on either protein or DNA synthesis. In contrast, in the presence of anti-laminin antibody, protein synthesis was decreased in a concentration-dependent manner, a significant effect being observed at the highest concentration after treatment for 24 h. At this concentration, DNA synthesis was also decreased after 48 h of culture, an effect that may be relevant to a hibernal reduction in claw cell turnover, and the associated seasonal vulnerability of cows to claw damage. The results provide evidence for basement membrane disruption at ulcer sites, and an increased potential for disruption in the diseased claw, and a causal link between this and abnormal epidermal keratinization. Basement membrane disruption is in turn associated with reciprocal changes in MMPs and their inhibitors, favouring extracellular proteolysis. Whether MMP activation is the primary cause of dermal-epidermal deterioration and, if so, how MMP activation is triggered, remains to be determined.

From the journals

Research continues apace into why some wounds fail to heal. Having identified that elevated levels of matrix metalloproteinases (MMPs) are often associated with recalcitrant healing, this Scandinavian study looked at the local profiles of MMP-9, MMP-2 and MMP-14 in normally healing skin wounds (acute) and chronic wounds of different aetiologies.

The role of matrix metalloproteinases in glioma invasion.

The matrix metalloproteinase (MMP) family plays an important role in the degradation of extracellular matrix (ECM) in various physiological and pathological conditions. Accumulated evidence has suggested that MMPs contribute to cancer cell invasion of the surrounding normal tissues and metastasis through the cell-surface ECM degradation. Strong correlations have been reported between elevated MMP levels and tumor cell invasiveness in human gliomas. Among them, attention has been focused on gelatinases (MMP-2 and MMP-9) and membrane type MMPs (MT-MMPs). We discuss here the biological significance of these MMPs in the glioblastoma invasion processes. A better understanding of cell-ECM interactions will help in developing therapeutic strategies to decrease the invasion of gliomas.