Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|B, Eq, H, M, Po, R, Rb||ELISA, IH(P), WB||M||Purified||Monoclonal Antibody|
|Presentation||Liquid in 0.1 M sodium phosphate buffer, pH 7.0 containing 2% protease free bovine Serum albumin.|
|Safety Information according to GHS|
|Material Size||100 µg|
References | 45 Available | See All References
|Reference overview||Pub Med ID|
|Folate deficiency decreases apoptosis of endometrium decidual cells in pregnant mice via the mitochondrial pathway. |
Liao, XG; Li, YL; Gao, RF; Geng, YQ; Chen, XM; Liu, XQ; Ding, YB; Mu, XY; Wang, YX; He, JL
Nutrients 7 1916-32 2015
It is well known that maternal folate deficiency results in adverse pregnancy outcomes. In addition to aspects in embryonic development, maternal uterine receptivity and the decidualization of stromal cells is also very important for a successful pregnancy. In this study, we focused on endometrium decidualization and investigated whether apoptosis, which is essential for decidualization, was impaired. Flow cytometry and TUNEL detection revealed that apoptosis of mouse endometrium decidual cells was suppressed in the dietary folate-deficient group on Days 7 and 8 of pregnancy (Day 1 = vaginal plug) when decidua regression is initiated. The endometrium decidual tissue of the folate deficiency group expressed less Bax compared to the normal diet group while they had nearly equal expression of Bcl2 protein. Further examination revealed that the mitochondrial transmembrane potential (ΔΨm) decreased, and the fluorescence of diffuse cytoplasmic cytochrome c protein was detected using laser confocal microscopy in normal decidual cells. However, no corresponding changes were observed in the folate-deficient group. Western blotting analyses confirmed that more cytochrome c was released from mitochondria in normal decidual cells. Taken together, these results demonstrated that folate deficiency could inhibit apoptosis of decidual cells via the mitochondrial apoptosis pathway, thereby restraining decidualization of the endometrium and further impairing pregnancy.
|Inhibition of SHP2 in basal-like and triple-negative breast cells induces basal-to-luminal transition, hormone dependency, and sensitivity to anti-hormone treatment. |
Zhao, H; Agazie, YM
BMC cancer 15 109 2015
The Src homology phosphotyrosyl phosphatase 2 (SHP2) is a positive effector of cell growth and survival signaling as well transformation induced by multiple tyrosine kinase oncogenes. Since the basal-like and triple-negative breast cancer (BTBC) is characterized by dysregulation of multiple tyrosine kinase oncogenes, we wanted to determine the importance of SHP2 in BTBC cell lines.Short hairpin RNA-based and dominant-negative expression-based SHP2 inhibition techniques were used to interrogate the functional importance of SHP2 in BTBC cell biology. In addition, cell viability and proliferation assays were used to determine hormone dependency for growth and sensitivity to anti-estrogen treatment.We show that inhibition of SHP2 in BTBC cells induces luminal-like epithelial morphology while suppressing the mesenchymal and invasive property. We have termed this process as basal-to-luminal transition (BLT). The occurrence of BLT was confirmed by the loss of the basal marker alpha smooth muscle actin and the acquisition of the luminal marker cytokeratin 18 (CK18) expression. Furthermore, the occurrence of BLT led to estrogen receptor alpha (ERα) expression, hormone dependency, and sensitivity to tamoxifen treatment.Our data show that inhibition of SHP2 induces BLT, ERα expression, dependency on estrogen for growth, and sensitivity to anti-hormone therapy. Therefore, inhibition of SHP2 may provide a therapeutic benefit in basal-like and triple-negative breast cancer.
|Masseter muscle myofibrillar protein synthesis and degradation in an experimental critical illness myopathy model. |
Akkad, H; Corpeno, R; Larsson, L
PloS one 9 e92622 2014
Critical illness myopathy (CIM) is a debilitating common consequence of modern intensive care, characterized by severe muscle wasting, weakness and a decreased myosin/actin (M/A) ratio. Limb/trunk muscles are primarily affected by this myopathy while cranial nerve innervated muscles are spared or less affected, but the mechanisms underlying these muscle-specific differences remain unknown. In this time-resolved study, the cranial nerve innervated masseter muscle was studied in a unique experimental rat intensive care unit (ICU) model, where animals were exposed to sedation, neuromuscular blockade (NMB), mechanical ventilation, and immobilization for durations varying between 6 h and 14d. Gel electrophoresis, immunoblotting, RT-PCR and morphological staining techniques were used to analyze M/A ratios, myofiber size, synthesis and degradation of myofibrillar proteins, and levels of heat shock proteins (HSPs). Results obtained in the masseter muscle were compared with previous observations in experimental and clinical studies of limb muscles. Significant muscle-specific differences were observed, i.e., in the masseter, the decline in M/A ratio and muscle fiber size was small and delayed. Furthermore, transcriptional regulation of myosin and actin synthesis was maintained, and Akt phosphorylation was only briefly reduced. In studied degradation pathways, only mRNA, but not protein levels of MuRF1, atrogin-1 and the autophagy marker LC3b were activated by the ICU condition. The matrix metalloproteinase MMP-2 was inhibited and protective HSPs were up-regulated early. These results confirm that the cranial nerve innervated masticatory muscles is less affected by the ICU-stress response than limb muscles, in accordance with clinical observation in ICU patients with CIM, supporting the model' credibility as a valid CIM model.
|Endothelial nitric oxide synthase and superoxide mediate hemodynamic initiation of intracranial aneurysms. |
Liaw, N; Fox, JM; Siddiqui, AH; Meng, H; Kolega, J
PloS one 9 e101721 2014
Hemodynamic insults at arterial bifurcations are believed to play a critical role in initiating intracranial aneurysms. Recent studies in a rabbit model indicate that aneurysmal damage initiates under specific wall shear stress conditions when smooth muscle cells (SMCs) become pro-inflammatory and produce matrix metalloproteinases (MMPs). The mechanisms leading to SMC activation and MMP production during hemodynamic aneurysm initiation are unknown. The goal is to determine if nitric oxide and/or superoxide induce SMC changes, MMP production and aneurysmal remodeling following hemodynamic insult.Bilateral common carotid artery ligation was performed on rabbits (n = 19, plus 5 sham operations) to induce aneurysmal damage at the basilar terminus. Ligated animals were treated with the nitric oxide synthase (NOS) inhibitor LNAME (n = 7) or the superoxide scavenger TEMPOL (n = 5) and compared to untreated animals (n = 7). Aneurysm development was assessed histologically 5 days after ligation. Changes in NOS isoforms, peroxynitrite, reactive oxygen species (ROS), MMP-2, MMP-9, and smooth muscle α-actin were analyzed by immunohistochemistry.LNAME attenuated ligation-induced IEL loss, media thinning and bulge formation. In untreated animals, immunofluorescence showed increased endothelial NOS (eNOS) after ligation, but no change in inducible or neuronal NOS. Furthermore, during aneurysm initiation ROS increased in the media, but not the intima, and there was no change in peroxynitrite. In LNAME-treated animals, ROS production did not change. Together, this suggests that eNOS is important for aneurysm initiation but not by producing superoxide. TEMPOL treatment reduced aneurysm development, indicating that the increased medial superoxide is also necessary for aneurysm initiation. LNAME and TEMPOL treatment in ligated animals restored α-actin and decreased MMPs, suggesting that eNOS and superoxide both lead to SMC de-differentiation and MMP production.Aneurysm-inducing hemodynamics lead to increased eNOS and superoxide, which both affect SMC phenotype, increasing MMP production and aneurysmal damage.
|Histological, histochemical, and protein changes after induced malocclusion by occlusion alteration of Wistar rats. |
Guerra, Cde S; Carla Lara Pereira, Y; Issa, JP; Luiz, KG; Guimarães, EA; Gerlach, RF; Iyomasa, MM
BioMed research international 2014 563463 2014
Although disorders of the stomatognathic system are common, the mechanisms involved are unknown. Our objective was to study the changes in the masseter muscles after unilateral exodontia. Molar extraction was performed on Wistar rats (left side), and the animals were sacrificed after either 14 or 26 days. The masseter muscle was processed for histological analysis, conventional and in situ zymography, and immunohistochemistry. The morphological analysis showed unique and specific characteristics for the experimental group. By conventional zymography no significant values of 72 kDa MMP-2 (P less than 0.05) were found in both of the sides of masseter muscle after 14 and 26 days of unilateral extraction. The in situ zymography showed gelatinolytic activity on all deep masseter muscles, with significant increase on the contralateral side after 14 and 26 days (P less than 0.05). The immunohistochemistry demonstrated greater expression of MMP-2 than MMP-9 and MMP-14 in all masseter muscles and there were few differences in the staining of 4 TIMPs. This knowledge about morphology and molecular masticatory muscle remodeling following environmental interventions can be used to develop clinically successful treatments.
|Differential effects of caveolin-1 and -2 knockdown on aqueous outflow and altered extracellular matrix turnover in caveolin-silenced trabecular meshwork cells. |
Aga, M; Bradley, JM; Wanchu, R; Yang, YF; Acott, TS; Keller, KE
Investigative ophthalmology & visual science 55 5497-509 2014
A single nucleotide polymorphism (SNP) identified between caveolin-1 (CAV1) and caveolin-2 (CAV2) on chromosome 7 is associated with glaucoma. One function of CAVs is endocytosis and recycling of extracellular matrix (ECM) components. Here, we generated CAV-silencing lentivirus to evaluate the effects on ECM turnover by trabecular meshwork (TM) cells and to measure the effect on outflow facility in anterior segment perfusion culture.Short hairpin CAV1 and CAV2 silencing and control lentivirus were generated, characterized, and applied to anterior segments in perfusion culture. Colocalization of CAVs with various ECM molecules in TM cells was investigated using immunofluorescence and confocal microscopy. Western immunoblotting and fluorogenic-based enzyme activity assays were used to investigate ECM protein levels and degradation, respectively.Endogenous CAVs colocalized with cortactin at podosome- or invadopodia-like structures (PILS), which are areas of focal ECM degradation. In perfusion culture, outflow rates increased significantly in CAV1-silenced anterior segments, whereas outflow significantly decreased in CAV2-silenced anterior segments. Matrix metalloproteinase (MMP)2 and MMP14, and a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS4) colocalized with both CAVs in TM cells. Protein levels and enzyme activities of MMP/ADAMTS4, fibronectin protein levels, actin stress fibers, and α-smooth muscle actin were all increased in CAV-silenced cells.Caveolin-mediated endocytosis is one mechanism by which TM cells can alter the physiological catabolism of ECM in order to change the composition of the outflow channels in the TM to regulate aqueous outflow resistance. Dysregulation of CAV function could contribute to the pathological changes in ECM that are observed in glaucoma.
|Role of Cox-2 in vascular inflammation: an experimental model of metabolic syndrome. |
Renna, NF; Diez, ER; Lembo, C; Miatello, RM
Mediators of inflammation 2013 513251 2013
The objective of this work was to demonstrate the role of COX-2 enzyme at the vascular in experimental model of metabolic syndrome. SHR male WKY rats were employed; they were distributed in 8 groups (n = 8 each): control (W); W + L: WKY rats receiving 20 mg/kg of lumiracoxib by intraesophageal administration; SHR; SHR + L: SHR + 20 mg/kg of lumiracoxib by intraesophageal administration; Fructose-Fed Rats (FFR): WKY rats receiving 10% (w/v) fructose solution in drinking water during all 12 weeks; FFR + L: FFR + 20 mg/kg of lumiracoxib by intraesophageal administration; Fructose-Fed Hypertensive Rats (FFHR): SHR receiving 10% (w/v) fructose solution in drinking water during all 12 weeks; and FFHR + L: FFHR + 20 mg/kg of lumiracoxib by intraesophageal administration. Metabolic variables, blood pressure, morphometric variables, and oxidative stress variables were evaluated; also MMP-2 and MMP-9 (collagenases), VCAM-1, and NF- κ B by Westernblot or IFI were evaluated. FFHR presented all variables of metabolic syndrome; there was also an increase in oxidative stress variables; vascular remodeling and left ventricular hypertrophy were evidenced along with a significant increase in the expression of the mentioned proinflammatory molecules and increased activity and expression of collagenase. Lumiracoxib was able to reverse vascular remodeling changes and inflammation, demonstrating the involvement of COX-2 in the pathophysiology of vascular remodeling in this experimental model.
|Expression of MMP-2 and MMP-9 in the rat trigeminal ganglion during the development of temporomandibular joint inflammation. |
Nascimento, GC; Rizzi, E; Gerlach, RF; Leite-Panissi, CR
Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas / Sociedade Brasileira de Biofisica ... [et al.] 46 956-967 2013
Orofacial pain is a prevalent symptom in modern society. Some musculoskeletal orofacial pain is caused by temporomandibular disorders (TMDs). This condition has a multi-factorial etiology, including emotional factors and alteration of the masticator muscle and temporomandibular joints (TMJs). TMJ inflammation is considered to be a cause of pain in patients with TMD. Extracellular proteolytic enzymes, specifically the matrix metalloproteinases (MMPs), have been shown to modulate inflammation and pain. The purpose of this investigation was to determine whether the expression and level of gelatinolytic activity of MMP-2 and MMP-9 in the trigeminal ganglion are altered during different stages of temporomandibular inflammation, as determined by gelatin zymography. This study also evaluated whether mechanical allodynia and orofacial hyperalgesia, induced by the injection of complete Freund's adjuvant into the TMJ capsule, were altered by an MMP inhibitor (doxycycline, DOX). TMJ inflammation was measured by plasma extravasation in the periarticular tissue (Evans blue test) and infiltration of polymorphonuclear neutrophils into the synovial fluid (myeloperoxidase enzyme quantification). MMP expression in the trigeminal ganglion was shown to vary during the phases of the inflammatory process. MMP-9 regulated the early phase and MMP-2 participated in the late phase of this process. Furthermore, increases in plasma extravasation in periarticular tissue and myeloperoxidase activity in the joint tissue, which occurred throughout the inflammation process, were diminished by treatment with DOX, a nonspecific MMP inhibitor. Additionally, the increases of mechanical allodynia and orofacial hyperalgesia were attenuated by the same treatment.
|Expression of soluble and functional full-length human matrix metalloproteinase-2 in Escherichia coli. |
Gonçalves, AN; Meschiari, CA; Stetler-Stevenson, WG; Nonato, MC; Alves, CP; Espreafico, EM; Gerlach, RF
Journal of biotechnology 157 20-4 2012
Characterization of the matrix metalloproteinase-2 (MMP-2) substrates and understanding of its function remain difficult because up to date preparations containing minor amounts of other eukaryotic proteins that are co-purified with MMP-2 are still used. In this work, the expression of a soluble and functional full-length recombinant human MMP-2 (rhMMP-2) in the cytoplasm of Escherichia coli is reported, and the purification of this metalloproteinase is described. Culture of this bacterium at 18°C culminated in maintenance of the soluble and functional rhMMP-2 in the soluble fraction of the E. coli lysate and its purification by affinity with gelatin-sepharose yielded approximately 0.12mg/L of medium. Western Blotting and zymographic analysis revealed that the most abundant form was the 72-kDa MMP-2, but some gelatinolytic bands corresponding to proteins with lower molecular weight were also detected. The obtained rhMMP-2 was demonstrated to be functional in a gelatinolytic fluorimetric assay, suggesting that the purified rhMMP-2 was correctly folded. The method described here involves fewer steps, is less expensive, and is less prone to contamination with other proteinases and MMP inhibitors as compared to expression of rhMMP-2 in eukaryotic tissue culture. This protocol will facilitate the use of the full-length rhMMP-2 expressed in bacteria and will certainly help researchers to acquire new knowledge about the substrates and biological activities of this important proteinase.
|MMP2 and acrosin are major proteinases associated with the inner acrosomal membrane and may cooperate in sperm penetration of the zona pellucida during fertilization. |
Ferrer, M; Rodriguez, H; Zara, L; Yu, Y; Xu, W; Oko, R
Cell and tissue research 349 881-95 2012
Sperm-zona pellucida (ZP) penetration during fertilization is a process that most likely involves enzymatic digestion of this extracellular coat by spermatozoa. Since the inner acrosomal membrane (IAM) is the leading edge of spermatozoa during penetration and proteins required for secondary binding of sperm to the zona are present on it, the IAM is the likely location of these enzymes. The objectives of this study were to identify and characterize proteinases present on the IAM, confirm their localization and provide evidence for their role in fertilization. Gelatin zymography of detergent extracts of the IAM revealed bands of enzymatic activity identified as serine and matrix metallo-proteinases (MMPs). Specific inhibitors to MMPs revealed that MMP activity was due to MMP2. Immunoblotting determined that the serine protease activity on the zymogram was due to acrosin and also confirmed the MMP2 activity. Immunogold labeling of spermatozoa at the electron microscope level showed that acrosin and MMP2 were confined to the apical and principal segments of the acrosome in association with the IAM, confirming our IAM isolation technique. Immunohistochemical examination of acrosin and MMP2 during spermiogenesis showed that both proteins originate in the acrosomic granule during the Golgi phase and later redistribute to the acrosomal membrane. Anti-MMP2 antibodies and inhibitors incorporated into in vitro fertilization media significantly decreased fertilization rates. This is the first study to demonstrate that MMP2 and acrosin are associated with the IAM and introduces the possibility of their cooperation in enzymatic digestion of the ZP during penetration.
|HPV16 oncoproteins induce MMPs/RECK-TIMP-2 imbalance in primary keratinocytes: possible implications in cervical carcinogenesis. |
Cardeal, LB; Boccardo, E; Termini, L; Rabachini, T; Andreoli, MA; di Loreto, C; Longatto Filho, A; Villa, LL; Maria-Engler, SS
PloS one 7 e33585 2012
Cervical cancer is the third most common cancer in women worldwide. Persistent infection with high-risk HPV types, principally HPV16 and 18 is the main risk factor for the development of this malignancy. However, the onset of invasive tumor occurs many years after initial exposure in a minority of infected women. This suggests that other factors beyond viral infection are necessary for tumor establishment and progression. Tumor progression is characterized by an increase in secretion and activation of matrix metalloproteinases (MMPs) produced by either the tumor cells themselves or tumor-associated fibroblasts or macrophages. Increased MMPs expression, including MMP-2, MMP-9 and MT1-MMP, has been observed during cervical carcinoma progression. These proteins have been associated with degradation of ECM components, tumor invasion, metastasis and recurrence. However, few studies have evaluated the interplay between HPV infection and the expression and activity of MMPs and their regulators in cervical cancer. We analyzed the effect of HPV16 oncoproteins on the expression and activity of MMP-2, MMP-9, MT1-MMP, and their inhibitors TIMP-2 and RECK in cultures of human keratinocytes. We observed that E7 expression is associated with increased pro-MMP-9 activity in the epithelial component of organotypic cultures, while E6 and E7 oncoproteins co-expression down-regulates RECK and TIMP-2 levels in organotypic and monolayers cultures. Finally, a study conducted in human cervical tissues showed a decrease in RECK expression levels in precancer and cancer lesions. Our results indicate that HPV oncoproteins promote MMPs/RECK-TIMP-2 imbalance which may be involved in HPV-associated lesions outcome.
|Angiotensin-II and rosuvastatin influence matrix remodeling in human mesangial cells via metalloproteinase modulation. |
Solini A, Rossi C, Santini E, Madec S, Salvati A, Ferrannini E.
Journal of hypertension 29 1930-9 2011
Persistent inflammation and oxidative stress influence the progression of diabetic nephropathy. Metalloproteinases (MMPs) participate in extracellular matrix remodeling. Statins show favorable anti-inflammatory effects in chronic kidney disease. We evaluated the effect of rosuvastatin on inflammatory and pro-fibrotic responses due to exposure to different glucose or free fatty acid (FFA) concentrations.
|Tissue inhibitor of metalloproteinase-2 gene delivery ameliorates postinfarction cardiac remodeling. |
Ramani, R; Nilles, K; Gibson, G; Burkhead, B; Mathier, M; McNamara, D; McTiernan, CF
Clinical and translational science 4 24-31 2011
Adenoviral-mediated (AdV-T2) overexpression of TIMP-2 would blunt ventricular remodeling and improve survival in a murine model of chronic ischemic injury.Male mice (n = 124) aged 10-14 weeks underwent either (1) left coronary artery ligation to induce myocardial infarction (MI group, n = 36), (2) myocardial injection of 6 × 10¹⁰ viral particles of AdV-T2 immediately post-MI (MI + T2 group, n = 30), (3) myocardial injection of 6 × 10¹⁰ viral particles of a control adenovirus (MI + Ct, n = 38), or 4) received no intervention (controls, n = 20). On post-MI day 7, surviving mice (n = 79) underwent echocardiographic, immunohistochemical, and biochemical analysis.In infarcted animals, the MI + T2 group demonstrated improved survival (p less than 0.02), better preservation of developed pressure and ventricular diameter (p less than 0.04), and the lowest expression and activity of MMP-2 and MMP-9 (p less than 0.04) compared with MI and MI + Ct groups. All infarcted hearts displayed significantly increased inflammatory cell infiltration (p less than 0.04 vs. control, MI, or MI + T2), with infiltration highest in the MI + Ct group and lowest in the MI + T2 group (p less than 0.04).Adenoviral mediated myocardial delivery of the TIMP-2 gene improves post-MI survival and limits adverse remodeling in a murine model of MI.
|MicroRNA-29b suppresses tumor angiogenesis, invasion, and metastasis by regulating matrix metalloproteinase 2 expression. |
Fang JH, Zhou HC, Zeng C, Yang J, Liu Y, Huang X, Zhang JP, Guan XY, Zhuang SM
Hepatology (Baltimore, Md) 54 1729-40. doi 2011
Hepatocellular carcinoma (HCC) is a highly vascularized tumor with frequent intrahepatic metastasis. Active angiogenesis and metastasis are responsible for rapid recurrence and poor survival of HCC. We previously found that microRNA-29b (miR-29b) down-regulation was significantly associated with poor recurrence-free survival of HCC patients. Therefore, the role of miR-29b in tumor angiogenesis, invasion, and metastasis was further investigated in this study using in vitro capillary tube formation and transwell assays, in vivo subcutaneous and orthotopic xenograft mouse models, and Matrigel plug assay, and human HCC samples. Both gain- and loss-of-function studies showed that miR-29b dramatically suppressed the ability of HCC cells to promote capillary tube formation of endothelial cells and to invade extracellular matrix gel in vitro. Using mouse models, we revealed that tumors derived from miR-29b-expressed HCC cells displayed significant reduction in microvessel density and in intrahepatic metastatic capacity compared with those from the control group. Subsequent investigations revealed that matrix metalloproteinase-2 (MMP-2) was a direct target of miR-29b. The blocking of MMP-2 by neutralizing antibody or RNA interference phenocopied the antiangiogenesis and antiinvasion effects of miR-29b, whereas introduction of MMP-2 antagonized the function of miR-29b. We further disclosed that miR-29b exerted its antiangiogenesis function, at least partly, by suppressing MMP-2 expression in tumor cells and, in turn, impairing vascular endothelial growth factor receptor 2-signaling in endothelial cells. Consistently, in human HCC tissues and mouse xenograft tumors miR-29b level was inversely correlated with MMP-2 expression, as well as tumor angiogenesis, venous invasion, and metastasis. Conclusion: miR-29b deregulation contributes to angiogenesis, invasion, and metastasis of HCC. Restoration of miR-29b represents a promising new strategy in anti-HCC therapy. (HEPATOLOGY 2011;).Copyright © 2011 American Association for the Study of Liver Diseases.
|Tempol inhibits TGF-β and MMPs upregulation and prevents cardiac hypertensive changes. |
Rizzi E, Castro MM, Ceron CS, Neto-Neves EM, Prado CM, Rossi MA, Tanus-Santos JE, Gerlach RF
International journal of cardiology 2011
|Matrix metalloproteinases 2 and 9 in the cochlea: expression and activity after aminoglycoside exposition. |
Setz C, Brand Y, Radojevic V, Hanusek C, Mullen PJ, Levano S, Listyo A, Bodmer D
The matrix metalloproteinases (MMPs) are a family of proteins involved in the remodelling and homeostasis of the extracellular matrix. These proteases have been well studied in the retina and the brain, marking their importance in neuronal cell survival and death [Chintala (2006) Exp Eye Res 82:5-12; Candelario-Jalil et al. (2009) Neuroscience 158:983-994]. The neuroepithelia of the eye and the inner ear share common characteristics. Therefore, we hypothesized that MMPs could play a similar role in the cochlea as described in the retina. We focused on the localization and function of MMP-2 and MMP-9 in the cochlea, by determining their expression and activity under normal conditions and after cochlear damage via aminoglycoside exposition. We examined their expression in 5-day-old Wistar rat cochleas by RT-PCR, real-time PCR, and Western blot. We used immunohistochemistry to investigate their location in the cochleas of adult C57BL/6 mice. We also determined whether or not the exposure of the organs of Corti to aminoglycosides would change MMP-2 and MMP-9 expression patterns. Western blotting identified MMP-2 and MMP-9 in neonatal spiral ganglion, stria vascularis, and to a lesser extent the organ of Corti. Neonatal mRNA expression of MMP-2 was approximately equivalent in all three tissues, while MMP-9 mRNA was highest in spiral ganglion. Immunohistochemistry showed MMP-2 primarily in adult spiral ganglion neurons and inner hair cells, while MMP-9 was found mainly in spiral ganglion neurons, inner hair cells and supporting cells. Organs of Corti treated with gentamicin for 24 h showed an upregulation of MMP-2 and MMP-9 proteins, but did not show a significant upregulation of mRNA expression 3, 6, 12, 24, and 36 h after gentamicin exposure. Inhibition of MMP activity in organs of Corti incubated with an MMP inhibitor in organotypic cultures resulted in hair cell death-suggesting that a basal level of MMP activity is required for hair cell survival.Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.
|Lymphocytic infiltration leads to degradation of lacrimal gland extracellular matrix structures in NOD mice exhibiting a Sjögren's syndrome-like exocrinopathy. |
Schenke-Layland, K; Xie, J; Magnusson, M; Angelis, E; Li, X; Wu, K; Reinhardt, DP; Maclellan, WR; Hamm-Alvarez, SF
Experimental eye research 90 223-37 2010
We previously reported that lacrimal glands (LGs) of male non-obese diabetic (NOD) mice, an established mouse model of autoimmune inflammatory LG disease that displays many features of human LGs in patients afflicted with Sjögren's syndrome (SjS), exhibit significant degradation of extracellular matrix (ECM) structures as well as increased expression of matrix metalloproteinases (MMPs). The purpose of the current study was to expand the spectrum of proteases identified, to clarify their probable origin as well as to identify the contribution of these changes to disease pathogenesis. We explored in depth the changes in ECM structures and ECM protease expression at the onset of disease (6 weeks) versus late stage disease (18 weeks) in male NOD mouse LGs, relative to LGs of age-matched male NODscid, a severely immunocompromised congenic strain, and healthy BALB/c mice. LG tissues were examined using routine histological, immunohistochemical, Western Blot and gene expression analyses novel multiphoton imaging technologies. We further characterized the profile of infiltrating immune cells under each condition using flow cytometry. Our results show that the initial infiltrating cells at 6 weeks of age are responsible for increased MMP and cathepsin H expression and therefore initiate the LG ECM degradation in NOD mice. More importantly, NODscid mice exhibited normal LG ECM structures, indicating the lymphocytes seen in the LGs of NOD mice are responsible for the degradation of the LG ECM. The disease-related remodeling of LG ECM structures may play a crucial role in altering the acinar signaling environment, disrupting the signaling scaffolds within the cells, which are required to mobilize the exocytotic trafficking machinery, ultimately leading to a loss of LG function in patients afflicted with SjS.Full Text Article
|Spironolactone and hydrochlorothiazide exert antioxidant effects and reduce vascular matrix metalloproteinase-2 activity and expression in a model of renovascular hypertension. |
Ceron CS, Castro MM, Rizzi E, Montenegro MF, Fontana V, Salgado MC, Gerlach RF, Tanus-Santos JE
Br J Pharmacol 160 77-87. Epub 2010 Mar 19. 2010
BACKGROUND AND PURPOSE: Increased oxidative stress and up-regulation of matrix metalloproteinases (MMPs) may cause structural and functional vascular changes in renovascular hypertension. We examined whether treatment with spironolactone (SPRL), hydrochlorothiazide (HCTZ) or both drugs together modified hypertension-induced changes in arterial blood pressure, aortic remodelling, vascular reactivity, oxidative stress and MMP levels and activity, in a model of renovascular hypertension.Full Text Article
|Molecular changes following topical photodynamic therapy using methyl aminolaevulinate in mouse skin. |
Choi JY, Park GT, Na EY, Wi HS, Lee SC, Lee JB
J Dermatol Sci 58 198-203. Epub 2010 Apr 4. 2010
BACKGROUND: Photodynamic therapy (PDT) with aminolevulinic acid (ALA) or methyl aminolaevulinate (MAL) has been shown to enhance treatment of photoaged skin. However, there is little information about the molecular changes involved in dermal matrix remodeling following MAL-PDT for photorejuvenation.
|Matrix metalloproteinase expression in teeth with apical periodontitis is differentially modulated by the modality of root canal treatment. |
Paula-Silva FW, da Silva LA, Kapila YL
J Endod 36 231-7. 2010
INTRODUCTION: The objective of this study was to investigate the expression of matrix metalloproteinases (MMPs) in apical periodontitis and during the periapical healing phase after root canal treatment.Full Text Article
|PPARgamma inhibits inflammatory reaction in oxidative stress induced human diploid fibloblast. |
Young-Hee Lee,Nan-Hee Lee,Govinda Bhattarai,Ji-Soo Yun,Tae-Il Kim,Eun-Chung Jhee,Ho-Keun Yi
Cell biochemistry and function 28 2010
The ageing of an inevitable life function is an unavoidable regressive physical process. Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor family. PPARgamma plays an important role in regulating several metabolic pathways. Recently, PPARgamma has been implicated in inflammatory responses and age-related diseases. The aim of this study was to determine the anti-inflammatory reaction of PPARgamma in an induced ageing progress. The late passage of human diploid fibroblasts (HDF), an in vitro ageing model, reveals the biological index materials of ageing. Aged cells showed decreased PPARgamma expression and elevated levels of intracellular adhesion molecule-1 (ICAM-1), an inflammatory molecule. To induce the aged cell phenotype, the middle stage of HDF cells (PD31) were induced stress induced premature senescence (SIPS) with 200 microM H(2)O(2) for 2 h. SIPS-HDF cells showed high levels of ICAM-1, extracellular signal regulated kinase (ERK1/2) activity and matrix metallomatrix protease (MMP-2, -9) activity, and low levels of PPARgamma expression. A reconstitution of SIPS HDF cells with Ad/PPARgamma resulted in the downregulation of ICAM-1, ERK1/2, MMP-2 and -9, and normalized growth of SIPS-HDF cells. Moreover, PPARgamma in aged HDF cells reduced pro-inflammatory molecules and eliminated the formation of reactive oxygen species (ROS) through the ERK1/2 pathway. These results strongly suggest that PPARgamma plays a key role in age-related inflammation and may have clinical applications as a molecular target in the treatment of age-related inflammation.
|Alteration of matrix metalloproteinases in selective left ventricular adriamycin-induced cardiomyopathy in the pig. |
Goetzenich A, Hatam N, Zernecke A, Weber C, Czarnotta T, Autschbach R, Christiansen S
The Journal of heart and lung transplantation : the official 28 1087-1093 2009
INTRODUCTION: Anthracyclines are widely used in oncogenic therapy. Owing to their cardiotoxic side effects, their application is subdued to dose limitations. Many cardioprotective approaches have failed. This study examined the role of matrix metalloproteinases (MMP) in the remodeling process of extracellular matrix after treatment with doxorubicin (Adriamycin) as a toehold for a new therapeutic approach, for example, treatment with MMP inhibitors. METHODS: Severe heart failure was induced in 6 pigs by the repetitive intracoronary application of Adriamycin. Degree of dilatation and insufficiency were measured by echocardiography and hemodynamics. Before and after treatment, MMP activity (fluorogenic assay: MMP-1, MMP-2) and gene expression (reverse transcription-polymerase chain reaction [RT-PCR]: MMP-1, -2, -9; membrane type-1 matrix metalloproteinase, [MT1MMP]; tissue inhibitor of metalloproteinase 1 [TIMP-1]) were measured. Spatial distribution of MMP-1, MMP-2, and collagen were visualized in antibody-stained frozen sections. One-way analysis of variance was used for data analysis. RESULTS: Severe myocardial insufficiency (ejection fractions < 50% of baseline values) developed in all animals. No severe side effects were encountered. We found a strong activation of MMP-1 and MMP-2 in fluorogenic and PCR assays. RT-PCR revealed a significant activation of MMP-9 and MT1-MMP and a weaker induction of TIMP-1. Histology showed typical signs of myocardial fibrosis, with myocardial cell loss, collagen disorder, and vacuoles. CONCLUSION: We showed a strong transcriptional activation for several specific MMPs in Adriamycin-induced cardiac remodeling. Contrary to published data on myocardial infarction, early inhibitory therapy before myocardial injury is possible in Adriamycin-treated patients. Local application by our catheter-based system would additionally help to avoid systemic side effects.
|Antioxidant treatment reduces matrix metalloproteinase-2-induced vascular changes in renovascular hypertension. |
Michele M Castro,Elen Rizzi,Gerson J Rodrigues,Carla S Ceron,Lusiane M Bendhack,Raquel F Gerlach,Jose E Tanus-Santos
Free radical biology & medicine 46 2009
Mounting evidence indicates that structural and functional vascular changes associated with two-kidney, one-clip (2K-1C) hypertension result, at least in part, from altered activity of matrix metalloproteinases (MMPs). Because MMPs are upregulated by increased formation of reactive oxygen species (ROS), we hypothesized that antioxidant approaches could attenuate the increases in MMP-2 expression/activity and the vascular dysfunction and remodeling associated with 2K-1C hypertension. Sham-operated or 2K-1C hypertensive rats were treated with tempol 18 mg/kg/day or apocyanin 25 mg/kg/day (or vehicle). Systolic blood pressure was monitored weekly. After 8 weeks of treatment, aortic rings were isolated to assess endothelium-dependent and -independent relaxation. Quantitative morphometry of structural changes in the aortic wall was studied in hematoxylin/eosin sections. Aortic and systemic ROS levels were measured using dihydroethidine and thiobarbituric acid-reactive substances, respectively. Aortic MMP-2 levels and activity were determined by gelatin and in situ zymography, fluorimetry, and immunohistochemistry. Tempol and apocyanin attenuated 2K-1C hypertension (181+/-20.8 and 192+/-17.6 mm Hg, respectively, versus 213+/-18 mm Hg in hypertensive controls; both p<0.05) and prevented the reduction in endothelium-dependent vasorelaxation found in 2K-1C rats. Tempol, but not apocyanin (p>0.05), prevented the vascular remodeling found in 2K-1C rats (all p<0.01). Tempol was more effective than apocyanin in attenuating hypertension-induced increases in oxidative stress (both p<0.05), MMP-2 levels, and MMP-2 activity in hypertensive rats (all p<0.05). Our results suggest that antioxidant approaches decrease MMP-2 upregulation and attenuate the vascular dysfunction and remodeling during 2K-1C hypertension.
|Doxycycline causes regression of endometriotic implants: a rat model. |
Pinar Akkaya, Gogsen Onalan, Nihan Haberal, Nilufer Bayraktar, Baris Mülayim, Hulusi B Zeyneloglu, Pinar Akkaya, Gogsen Onalan, Nihan Haberal, Nilufer Bayraktar, Baris Mülayim, Hulusi B Zeyneloglu
Human reproduction (Oxford, England) 24 1900-8 2009
BACKGROUND: Doxycycline (Dox) has a number of non-antibiotic properties. One of them is the inhibition of matrix metalloproteinase (MMP) activity. The aim of this study was to assess the effects of Dox in a rat endometriosis model. METHODS: Endometriosis was surgically induced in 40 rats by transplanting of endometrial tissue. After 3 weeks, repeat laparotomies were performed to check the implants and the animals were randomized into four groups: Group I, low-dose Dox (5 mg/kg/day); Group II, high-dose Dox (40 mg/kg/day); Group III, leuprolide acetate 1 mg/kg single dose, s.c.; and Group VI (controls), no medication. The treatment, initiated on the day of surgery and continuing for 3 weeks, was administered to the study groups. Three weeks later, the rats were euthanized and the implants were evaluated morphologically and histologically for immunoreactivity of MMP-2 and -9, and interleukin-6 (IL-6) concentration in the peritoneal fluid was assayed. RESULTS: Treatment with leuprolide acetate, or high-dose or low-dose Dox caused significant decreases in the implant areas compared with the controls (P = 0.03, P = 0.006, and P = 0.001, respectively). IL-6 levels in peritoneal fluid decreased in Group I (P = 0.02) and Group III (P 0.05). MMP H scores were significantly lower in the group that received low-dose Dox in both epithelial and stromal MMP-2 and -9 immunostaining when compared with the control group [P = 0.048, P = 0.002, P = 0.007 and P = 0.002, respectively, MMP-2 (epithelia), MMP-2 (stroma), MMP-9 (epithelia) and MMP-9 (stroma)]. CONCLUSIONS: Low-dose Dox caused regression of endometriosis in this experimental rat model.
|Glycogen synthase kinase-3beta is activated by matrix metalloproteinase-2 mediated proteolysis in cardiomyoblasts. |
Kandasamy, Arulmozhi D and Schulz, Richard
Cardiovasc. Res., 83: 698-706 (2009) 2009
AIMS: Matrix metalloproteinase (MMP)-2 contributes to myocardial oxidative stress injury by degrading sarcomeric and cytoskeletal proteins in cardiomyocytes. Glycogen synthase kinase (GSK)-3beta is dysregulated during oxidative stress and is susceptible to proteolytic cleavage. Here we determined whether GSK-3beta is a MMP-2 substrate as a result of oxidative stress. METHODS AND RESULTS: MMP-2 and GSK-3beta were incubated and the cleavage fragments were identified by immunoblotting and silver stain. The intact protein and its primary cleavage fragment were subjected to trypsin digestion and the resultant peptides were analysed by LC-MS/MS. GSK-3beta kinase activity was measured using a peptide substrate and [gamma-(32)P]-ATP. Oxidative stress in H9c2 cardiomyoblasts was induced by H(2)O(2) and the levels and activities of MMP-2 and GSK-3beta were measured. Incubation of 47 kDa GSK-3beta with MMP-2 resulted in the time- and concentration-dependant cleavage of GSK-3beta as seen by appearance of an approximately 30 kDa fragment. MS analysis and Mascot database search yielded a peptide with an amino acid sequence of GSK-3beta lacking the N-terminal region. GSK-3beta kinase activity was significantly increased upon incubation with MMP-2 which was abrogated by the MMP inhibitor GM-6001. H(2)O(2) challenge of H9c2 cardiomyoblasts significantly increased the activity and level of MMP-2, reduced the level of GSK-3beta, and significantly increased GSK-3beta kinase activity. Both the loss of intact GSK-3beta and increase in its kinase activity were reduced with MMP inhibitors. MMP-2 pull-down assays in H9c2 cell lysates showed the association of MMP-2 with GSK-3beta. CONCLUSION: GSK-3beta may be a target of MMP-2 and its cleavage by MMP-2 enhances its kinase activity. MMP-2 may cleave off the N-terminal of GSK-3beta where the inhibitory phosphorylation of serine-9 occurs. MMP-2-mediated augmentation of GSK-3beta kinase activity may contribute to cardiac injury resulting from enhanced oxidative stress.
|High matrix metalloproteinase activity is a hallmark of periapical granulomas. |
de Paula-Silva FW, D'Silva NJ, da Silva LA, Kapila YL
J Endod 35 1234-42 2009
INTRODUCTION: The inability to distinguish periapical cysts from granulomas before performing root canal treatment leads to uncertainty in treatment outcomes because cysts have lower healing rates. Searching for differential expression of molecules within cysts or granulomas could provide information with regard to the identity of the lesion or suggest mechanistic differences that may form the basis for future therapeutic intervention. Thus, we investigated whether granulomas and cysts exhibit differential expression of extracellular matrix (ECM) molecules. METHODS: Human periapical granulomas, periapical cysts, and healthy periodontal ligament tissues were used to investigate the differential expression of ECM molecules by microarray analysis. Because matrix metalloproteinases (MMP) showed the highest differential expression in the microarray analysis, MMPs were further examined by in situ zymography and immunohistochemistry. Data were analyzed by using one-way analysis of variance followed by the Tukey test. RESULTS: We observed that cysts and granulomas differentially expressed several ECM molecules, especially those from the MMP family. Compared with cysts, granulomas exhibited higher MMP enzymatic activity in areas stained for MMP-9. These areas were composed of polymorphonuclear cells (PMNs) in contrast to cysts. Similarly, MMP-13 was expressed by a greater number of cells in granulomas compared with cysts. CONCLUSION: Our findings indicate that high enzymatic MMP activity in PMNs together with MMP-9 and MMP-13 stained cells could be a molecular signature of granulomas unlike periapical cysts.Full Text Article
|Increased degradation of extracellular matrix structures of lacrimal glands implicated in the pathogenesis of Sjögren's syndrome. |
Schenke-Layland, K; Xie, J; Angelis, E; Starcher, B; Wu, K; Riemann, I; MacLellan, WR; Hamm-Alvarez, SF
Matrix biology : journal of the International Society for Matrix Biology 27 53-66 2008
Lacrimal glands (LGs) of male non-obese diabetic (NOD) mice display many features of human LGs in patients afflicted with the autoimmune disease Sjögren's syndrome (SS), including the loss of secretory functions and a lymphocytic infiltration into the glands by 4 months of age. So far, research has mainly focused on the intracellular events that are involved in initiating LG dysfunction; however, the impact of SS on extracellular matrix (ECM) structures of the diseased LGs has not yet been determined. In this study we identified and compared LG ECM formation and integrity of age-matched male healthy (BALB/c) and diseased (NOD) mice. LG tissues were examined using routine histological, biochemical, immunohistochemical and gene expression analysis. Multiphoton imaging and second-harmonic generation (SHG) microscopy permitted the non-invasive analysis of major LG ECM structures including collagen- and elastin-containing fibers. Biochemical testing demonstrated a significant loss of collagen, glycosaminoglycans and desmosine in NOD LGs when compared to healthy BALB/c LGs. Immunohistochemical staining and gene expression analysis confirmed this disease-related alteration of LG ECM structures. Furthermore, laser-induced autofluorescence and SHG microscopy revealed dramatic changes in the structural organization of most collagenous and elastic fibers of the diseased LG tissues that were more pronounced than those displayed by histological analysis. Our results clearly show an enhanced degradation of ECM proteins accompanied by the severe disorganization and deformation of ECM structures of diseased LG tissues. These new insights into the involvement of ECM degradation in SS may lead to novel therapies for patients suffering from dry eye disease.
|Metalloproteinase inhibition ameliorates hypertension and prevents vascular dysfunction and remodeling in renovascular hypertensive rats. |
Michele M Castro, Elen Rizzi, Lívia Figueiredo-Lopes, Karla Fernandes, Lusiane M Bendhack, Dimitrius Leonardo Pitol, Raquel F Gerlach, Jose E Tanus-Santos
Atherosclerosis 198 320-31 2008
Altered activity of matrix metalloproteinases (MMPs) is implicated in the vascular remodeling of hypertension. We examined whether increased MMP-2 expression/activity plays a role in the vascular remodeling and dysfunction found in the two-kidney, one-clip (2K-1C) hypertension. Sham operated or 2K-1C hypertension rats were treated with doxycycline 30mg/(kgday) (or vehicle). Systolic blood pressure was monitored weekly. After 8 weeks of treatment, aortic rings were isolated to assess endothelium-dependent and independent relaxations. Quantitative morphometry of structural changes, collagen, and elastin contents in the aortic wall were studied in hematoxylin/eosin, Sirius Red, and Orceine stained aortic sections, respectively. Aortic MMP-2 levels were determined by gelatin zymography and aortic MMP-2 proteolytic activity was measured using DQ gelatin as the substrate after MMP-2 was captured by a specific antibody and immobilized on a microplate. Aortic MMP-2/tissue inhibitor of metalloproteinases (TIMP)-2 mRNA levels were determined by real time RT-PCR. Doxycycline attenuated 2K-1C hypertension (215+/-8mmHg versus 167+/-13mmHg in 2K-1C rats and 2K-1C+doxy rats, respectively; P0.01) and prevented the 35% reduction in endothelium-dependent vasorelaxation found in 2K-1C rats. Doxycycline prevented the increases in media thickness, and was associated with lower media/lumen and cross-sectional areas (all P0.01). Doxycycline also prevented excessive collagen and elastin deposition in the vascular wall. Increased MMP-2 and Pro-MMP-2 levels and MMP-2 activity were found in the aortas of 2K-1C rats (all P0.05). A 21-fold increase (P0.001) in the ratio of MMP-2/TIMP-2 mRNA expression was found in the 2K-1C group, whereas this ratio remained unaltered in 2K-1C+doxy rats. Our results suggest that MMP-2 plays a role in 2K-1C hypertension and its structural and functional vascular changes, which were attenuated by doxycycline.
|Big ET-1 processing into vasoactive peptides in arteries and veins. |
Stephanie W Watts, Keshari Thakali, Chuck Smark, Catherine Rondelli, Gregory D Fink, Stephanie W Watts, Keshari Thakali, Chuck Smark, Catherine Rondelli, Gregory D Fink, Stephanie W Watts, Keshari Thakali, Chuck Smark, Catherine Rondelli, Gregory D Fink, Stephanie W Watts, Keshari Thakali, Chuck Smark, Catherine Rondelli, Gregory D Fink
Vascular pharmacology 47 302-12 2007
The endothelin (ET) peptides are more potent in contracting veins than arteries. The precursor big ET-1 is metabolized by endothelin converting enzyme [ECE; to ET-1 (1-21)], matrix metalloproteases [MMPs; to ET-1 (1-32)] and chymase [to ET-1(1-31)]. We hypothesized that arteries and veins were differently dependent in conversion of big ET-1 to vasoconstrictors. Immunohistochemical, western, zymographic and isometric contractile assays in rat aorta and vena cava were used. Big ET-1 contracted aorta [60+/-17% phenylephrine contraction] but was more efficacious in vena cava [478+/-61% norepinephrine contraction]. ECE and its product ET-1(1-21) were detected in aorta and vena cava, and the ECE inhibitors phosphoramidon and CGS-26393 reduced big ET-1-induced contraction. ET-1 (1-32) contracted aorta and vena cava but inhibition of MMPs with minocycline or GM6001 did not reduce big ET-1-induced contraction; zymography confirmed active tissue MMPs. Aorta and vena cava contracted to the product of chymase, ET-1 (1-31). Chymase was detected in aorta and only weakly in vena cava. Inhibition of chymase (chymostatin, 100 muM) reduced arterial (19% control) but not venous constriction to big ET-1. These results suggest at least one potential significant difference - the role of chymase - in in vitro enzymatic processing of big ET-1 in arteries and veins.Full Text Article
|Matrix metalloproteinases contribute to endotoxin and interleukin-1beta induced vascular dysfunction. |
M M Lalu, J Cena, R Chowdhury, A Lam, R Schulz
British journal of pharmacology 149 31-42 2006
BACKGROUND AND PURPOSE: The acute vascular inflammatory dysfunction associated with endotoxaemia may reflect an imbalance between matrix metalloproteinases (MMPs) and their natural inhibitors (TIMPs), induced by the endotoxin. This possibility was tested in rat aortic tissue. EXPERIMENTAL APPROACHES: Tone induced by phenylephrine in aortic rings was measured after exposure in vitro to ambient lipopolysaccharide (LPS) or the proinflammatory cytokine interleukin-1beta (IL-1beta) for 6h, with or without MMP inhibitors (doxycycline or GM6001). Gelatinase and MMP activities, TIMP proteins and contractility were measured in aortae taken from rats 6h after receiving LPS in vivo. KEY RESULTS: Inhibition of MMP prevented the loss of phenylephrine-induced tone in aortic rings after LPS or IL-1beta. IL-1beta also increased release of MMP-2 activity from aortic tissue. In aortae exposed in vivo to LPS, net gelatinase, MMP-9 activities and TIMP-1 protein levels were increased, whereas TIMP-4 was reduced. These aortae were hypocontractile to both phenylephrine and KCl. Hypocontractility was partially reversed by doxycycline ex vivo. CONCLUSIONS AND IMPLICATIONS:MMP inhibitors ameliorate vascular hyporeactivity induced by either LPS or IL-1beta in vitro. LPS in vivo alters the balance between MMPs and TIMPs, contributing to vascular dysfunction which is partially reversed by MMP inhibitors. Vascular MMPs are activated as a result of LPS or IL-1beta-induced stress and contribute to the hyporeactivity of blood vessels to vasoconstrictors.Full Text Article
|Increased activity of matrix metalloproteinase 2 and 9 after hepatic radiofrequency ablation. |
Lars Frich, Kristin Bjørnland, Solveig Pettersen, Ole Petter F Clausen, Ivar P Gladhaug
The Journal of surgical research 135 297-304 2006
BACKGROUND: Radiofrequency (RF) ablation of hepatic metastases from colorectal cancer (CRC) is associated with a high rate of local and intrahepatic tumor recurrence. Matrix metalloproteinases (MMPs) play an important role in inflammation, tissue repair and tumor cell invasion and metastasis. MMP-2 and MMP-9 are associated with increased risk of recurrence and decreased survival in patients with colorectal cancer. The primary aim of the study was to determine if hepatic RF ablation increased MMP-2 and MMP-9 activity in the transition zone surrounding the coagulated hepatic tissue. MATERIALS AND METHODS: Twelve pigs were randomized to hepatic RF ablation with (n = 6) or without (n = 6) hepatic vascular occlusion (Pringle maneuver). Four days after ablation tissue specimens were collected from the transition zone surrounding coagulated hepatic tissue, and from normal hepatic parenchyma. MMP activity was quantified by gelatin zymography. Cellular localization of MMPs was determined by immunohistochemistry using antibodies against MMP-2, MMP-9, and the macrophage marker CD68. RESULTS: MMP-2 and MMP-9 activity was increased in the transition zone compared to normal hepatic parenchyma, with ratios of 3.0 (P = 0.005) and 2.6 (P = 0.001), respectively. Pringle maneuver did not influence MMP activity. MMP-2 and MMP-9 expression was localized to macrophages in the transition zone. CONCLUSIONS: Hepatic RF ablation is associated with increased expression of MMP-2 and MMP-9 in macrophages in the transition zone surrounding the coagulated hepatic parenchyma. These findings may contribute to the understanding of possible mechanisms for the high recurrence rates observed in patients after RF ablation of CRC hepatic metastases.
|Ischemia-induced cleavage of cadherins in NRK cells requires MT1-MMP (MMP-14). |
Covington, MD; Burghardt, RC; Parrish, AR
American journal of physiology. Renal physiology 290 F43-51 2006
Ischemia is a leading cause of acute renal failure (ARF), a disease associated with high morbidity and mortality. Disruption of intercellular adhesion in the proximal tubules is linked to ARF, although the molecular mechanism(s) remains unclear. Our previous studies showed that ischemia is associated with cadherin cleavage and loss in NRK cells, putatively due to a matrix metalloproteinase (MMP) (7). In the current studies, a MMP required for E-cadherin cleavage and N-cadherin loss was identified. Chemical inhibitors against a number of soluble MMPs (1, 2, 3, 8, 9) failed to completely attenuate ischemia-induced cadherin loss. Under ischemic conditions, there was an increase in active membrane-type (MT)1-MMP but a decrease in MMP-2 protein expression. Plating cells on fibronectin protected against ischemia-induced loss of cadherins and, interestingly, no increase in active MT1-MMP levels was seen in ischemic cells on fibronectin-coated dishes. In addition, L cells stably expressing E- (LE) or N-cadherin (LN), but lacking MT1-MMP expression, were resistant to ischemia-induced cadherin loss. The role of MT1-MMP in ischemia-induced cadherin loss was confirmed by either blocking MT1-MMP activity with a neutralizing antibody or expression with shRNA constructs which protected full-length E- and N-cadherin during ischemia. Using shRNA constructs to suppress MT1-MMP expression, ischemia-induced disruption of cadherin function was ablated, and cell-cell contacts were preserved. These results demonstrate that ischemia induces increased expression of active MT1-MMP and subsequent disruption of cadherin/catenin complexes, implying that MT1-MMP plays a role in ischemia-induced ARF.
|Ischaemia-reperfusion injury activates matrix metalloproteinases in the human heart. |
Lalu, MM; Pasini, E; Schulze, CJ; Ferrari-Vivaldi, M; Ferrari-Vivaldi, G; Bachetti, T; Schulz, R
European heart journal 26 27-35 2005
Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulate matrix remodelling in the heart and play a pivotal role in myocardial dysfunction immediately following ischaemia-reperfusion injury ex vivo in rats. We investigated the changes in MMPs and TIMPs in acute myocardial ischaemia-reperfusion injury in humans.Fifteen patients with stable angina undergoing coronary artery bypass graft surgery with cardiopulmonary bypass were enrolled. Left ventricular stroke work index was monitored prior to bypass and for 24 h following reperfusion. Left atrial biopsy samples were obtained at the start of bypass before cardioplegia and within 10 min after removal of the aortic cross-clamp. Plasma samples were collected from the radial artery and coronary sinus 1, 5, and 10 min following removal of the cross-clamp. In cardiac biopsies there was a marked increase in 72 kDa MMP-2 and 92 kDa MMP-9 activities, and a decrease in TIMP-1 upon reperfusion. Increased MMP activity correlated positively with cross-clamp duration and inversely with cardiac mechanical function 3 h following reperfusion. TIMP-1 correlated inversely with cross-clamp time and positively with cardiac mechanical function. Plasma samples revealed a significant increase in both 92 kDa MMP-9 and 64 kDa MMP-2 activities 1 min following removal of cross-clamp.Reperfusion following cardioplegia activates MMPs in the myocardium and plasma of patients undergoing coronary artery bypass grafting. This is the first correlation of MMP myocardial activity with cardiac function in humans. The early increase in MMP activity produces a proteolytic environment that may contribute to myocardial stunning injury in humans.
|Cleavage of fibromodulin in cartilage explants involves removal of the N-terminal tyrosine sulfate-rich region by proteolysis at a site that is sensitive to matrix metalloproteinase-13. |
Heathfield, TF; Onnerfjord, P; Dahlberg, L; Heinegård, D
The Journal of biological chemistry 279 6286-95 2004
Integrity of cartilage fails in joint disease. The current work aimed to identify candidate active proteinases in joint diseases using an in vitro model for cartilage degradation induced by interleukin-1. A critical event in the process of cartilage destruction in joint disease is the failure of the collagen fiber network to maintain integrity. Proteins binding to the surface of the fibers are likely early points of failure. Fibromodulin, a member of the leucine-rich repeat protein family, is one predominant protein in cartilage and is known for its roles in the formation of collagen fibrils and sustained interaction with these formed fibers. Cleavage removes the tyrosine sulfate-rich region in the N terminus of fibromodulin. Whereas fibromodulin bound to collagen in tissue was digested, purified fibromodulin was not cleaved. In contrast an N-terminal 10-kDa fragment, Gln19-Lys98, of the protein generated by Lys-C digestion contains the cleavage site and was a substrate cleaved by the enzyme in medium from stimulated cultures. In solution, digestion of this substrate with matrix metalloproteinase (MMP)-2, -9, -8, and -13 demonstrated that only MMP-13 was capable to efficiently cleave it. The cleavage product obtained after MMP-13 digestion was identical to that observed in cleaved fibromodulin from cartilage explant cultures stimulated with interleukin-1. MMP-13 treatment of fresh articular cartilage also produced the fragment under study. The elucidation of the enzyme responsible for such cleavage may lead to treatment modalities involving its selective inhibition for patients suffering from arthritis. The known structure of the fragments permits the generation of neo-epitope antibodies to the cleavage site, which can be used to detect ongoing cartilage degradation in patients with arthritic disease, an important adjunct in monitoring disease progression, active disease, and efficacy of treatment.
|Matrix metalloproteinase activities are altered in the heart and plasma during endotoxemia. |
Manoj M Lalu, Tamás Csont, Richard Schulz
Critical care medicine 32 1332-7 2004
OBJECTIVE: To investigate whether myocardial and plasma matrix metalloproteinase (MMP) activities are altered during endotoxemia. DESIGN: Prospective randomized, animal study. SETTING: University research laboratory. SUBJECTS: Male Sprague-Dawley rats, 250-300 g. INTERVENTIONS: Rats were administered either bacterial lipopolysaccharide (LPS) or vehicle (pyrogen-free water). Groups of LPS-administered animals were killed at 0.5, 1, 3, 6, 12, and 24 hrs postinjection. Vehicle injected animals were killed at 6 hrs. Blood pressure was recorded before kill. Heart and plasma samples were analyzed by gelatin zymography and immunoblot. MEASUREMENTS AND MAIN RESULTS: Blood pressure was significantly depressed at 3-24 hrs post-LPS injection; however, overt symptoms of endotoxemia and reduction in blood pressure were most significant 6-12 hrs post-LPS. Heart samples from control rats revealed MMP-2 activity but no MMP-9 activity. MMP-2 activity was significantly depressed when overt symptoms of endotoxemia peaked at 6-12 hrs. Plasma MMP-2 activity significantly decreased 3-12 hrs after LPS injection. This loss of activity was associated with a loss of MMP-2 protein. In contrast, plasma MMP-9 activities were rapidly elevated following LPS injection, peaking between 1 and 12 hrs. MMP-9 activity correlated inversely with blood pressure. CONCLUSIONS: Endotoxemia induced rapid changes in MMP activity in both the myocardium and plasma. An increase in circulating MMP-9 activity may contribute to endotoxemic cardiovascular dysfunction.
|Region- and type-specific induction of matrix metalloproteinases in post-myocardial infarction remodeling. |
Wilson, EM; Moainie, SL; Baskin, JM; Lowry, AS; Deschamps, AM; Mukherjee, R; Guy, TS; St John-Sutton, MG; Gorman, JH; Edmunds, LH; Gorman, RC; Spinale, FG
Circulation 107 2857-63 2003
Induction of matrix metalloproteinases (MMPs) contributes to adverse remodeling after myocardial infarction (MI). Whether a region- and type-specific distribution of MMPs occurs within the post-MI myocardium remained unknown.Ten sheep were instrumented with a sonomicrometry array to measure dimensions in 7 distinct regions corresponding to the remote, transition, and MI regions. Eight sheep served as reference controls. The relative abundance of representative MMP types and the tissue inhibitors of the MMPs (TIMPs) was quantified by immunoblotting. Segment length increased from baseline in the remote (24.9+/-5.4%), transition (18.0+/-2.9%), and MI (53.8+/-11.0%) regions at 8 weeks after MI (Pless than 0.05) and was greatest in the MI region (Pless than 0.05). Region- and type-specific changes in MMPs occurred after MI. For example, MMP-1 and MMP-9 abundance was unchanged in the remote, fell to 3+/-2% in the transition, and was undetectable in the MI region (Pless than 0.05). MMP-13, MMP-8, and MT1-MMP increased by greater than 300% in the transition and MI regions (Pless than 0.05). TIMP abundance decreased significantly in the transition region after MI and fell to undetectable levels within the MI region.The unique findings of this study were 2-fold. First, changes in regional geometry after MI were associated with changes in MMP levels. Second, a region-specific portfolio of MMPs was induced after MI and was accompanied by a decline in TIMP levels, indicative of a loss of MMP inhibitory control. Targeting the regional imbalance between specific MMPs and TIMPs within the post-MI myocardium holds therapeutic potential.
|Matrix metalloproteinase inhibitor BB-3103 unlike the serine proteinase inhibitor aprotinin abrogates epidermal healing of human skin wounds ex vivo. |
Mirastschijski, U; Impola, U; Karsdal, MA; Saarialho-Kere, U; Agren, MS
The Journal of investigative dermatology 118 55-64 2002
Several matrix metalloproteinases and serine proteinases are upregulated in migrating keratinocytes during cutaneous wound repair. Single cell culture studies indicate the necessity for matrix metalloproteinases but not for serine proteinases in keratinocyte locomotion. To account for epithelial-mesenchymal interactions, an ex vivo human skin wound model was used to investigate the contribution of matrix metalloproteinases and serine proteinases to wound healing by treatment with broad-spectrum inhibitors of matrix metalloproteinases (BB-3103) or serine proteinases (aprotinin). Human skin explants with circular 3 mm superficial defects were incubated in culture medium without (controls) or with the proteinase inhibitors for 7 d. BB-3103 abrogated epithelialization (p less than 0.001), whereas aprotinin-treated wounds and controls were covered with new epithelium. Lack of epithelialization was unlikely due to cytotoxicity because the matrix metalloproteinase inhibitor did neither influence viability of cultured epidermal keratinocytes nor apoptosis in wounds. Involvement of specific matrix metalloproteinases in epithelialization was analyzed by gelatin zymography, western blotting, immunohistochemistry, and in situ hybridization. Wound healing was accompanied by active matrix metalloproteinase-1 and increased active matrix metalloproteinase-2 but irrespectively of active matrix metalloproteinase-9. BB-3103 blocked activation of matrix metalloproteinase-2 and matrix metalloproteinase-9 but not of matrix metalloproteinase-1. Active matrix metalloproteinase-2 localized solely to the dermis, whereas matrix metalloproteinase-9 was consistently found in new epithelium. Membrane-type 1 matrix metalloproteinase was undetectable in wound keratinocytes. BB-3103 and aprotinin reduced tumor necrosis factor-alpha in media but did not appreciably alter amounts of other soluble regulators of matrix metalloproteinases and epithelialization. Our findings demonstrate that keratinocyte migration is associated with active matrix metalloproteinase-2 but occurs independently of serine proteinases and active matrix metalloproteinase-9 in fibrin-deficient skin wound healing.
|Matrix metalloproteinase-2 and -9 in bile as a marker of liver metastasis in colorectal cancer. |
N Okada,H Ishida,N Murata,D Hashimoto,Y Seyama,S Kubota
Biochemical and biophysical research communications 288 2001
Matrix metallproteinases (MMP)-2 and -9 are associated with cancer invasion and metastasis. MMP-2 and MMP-9 activities have never been assayed in bile. In the present study we investigated whether MMP-2 and -9 activities in the bile could be a marker for evaluation of liver metastasis in colorectal cancer. Fifty-three patients underwent colorectal resection for histologically verified adenocarcinoma. Twenty-six patients had colorectal cancer without liver metastasis and 27 patients had metastatic liver tumor. Six patients were studied as carcinoma-free control. MMP-2 and MMP-9 activities were assayed in bile using gelatin zymography and quantitated. Active MMP-2 activity of colorectal cancer with liver metastasis group (24.1 +/- 2.5 pixel count) was significantly higher than that of colorectal cancer without liver metastasis group (11.4 +/- 1.3 pixel count) (P < 0.001) or of control group (6.4 +/- 1.0 pixel count) (P < 0.001). Active MMP-9 was not detected in bile. ProMMP-9 activity of colorectal cancer with liver metastasis group (530.3 +/- 127.5 pixel count) was significantly higher than that of colorectal cancer without liver metastasis group (213.9 +/- 33.2 pixel count) (P = 0.008). This is the first report showing that the levels of active MMP-2 and proMMP-9 in bile were significantly higher in liver metastasis of colorectal cancer than in metastasis-free colorectal cancer. The results suggest that activities of active MMP-2 and proMMP-9 in the bile may be useful markers for predicting liver metastasis in colorectal cancer.
|Inhibitors of prostaglandin synthesis inhibit human prostate tumor cell invasiveness and reduce the release of matrix metalloproteinases. |
F A Attiga, P M Fernandez, A T Weeraratna, M J Manyak, S R Patierno
Cancer research 60 4629-37 2000
Eicosanoids modulate the interaction of tumor cells with various host components in cancer metastasis. Their synthesis involves the release of arachidonic acid (AA) from cellular phospholipids by phospholipase A2 (PLA2), followed by metabolism by cyclooxygenases (COXs) and lipooxygenases (LOXs). This study aimed to identify the pathway(s) of AA metabolism that are required for the invasion of prostate tumor cells. DU-145 and PC-3 human prostate cancer cell lines were used to test the effect of inhibitors of PLA2, COX, or LOX on the invasion of prostate tumor cells through Matrigel in vitro using the Boyden chamber assay and fibroblast-conditioned medium as the chemoattractant. We used nontoxic doses that did not inhibit simple cell motility and did not decrease clonogenic survival. All of the inhibitors caused a significant reduction in AA release from treated cells compared with control cells, which indicated that the treatments were biochemically active. Invasion through Matrigel was inhibited by the PLA2 inhibitor 4-bromophenacyl bromide (4-BPB), the general COX inhibitor ibuprofen (IB), and the highly selective COX-2 inhibitor NS398. Inhibition of cell invasiveness by 4-BPB (1.0 microM), IB (10.0 microM), and NS398 (10.0 microM) was reversed by the addition of prostaglandin E2 (PGE2). PGE2 alone, however, did not stimulate invasiveness, which suggests that its production is necessary for rendering the cells invasive-permissive but not sufficient for inducing invasiveness. In contrast, we found no significant inhibition of invasion of prostate tumor cells treated with esculetin (1.0 microM) or nordihydroguiaretic acid (1.0 microM), which are specific inhibitors of LOX. We also tested the effect of 4-BPB, IB, NS398, and esculetin on the secretion of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), as key enzymes in the proteolysis of Matrigel during invasion, using gelatin zymograms and Western blots. Cells that received 4-BPB, IB, or NS398, but not esculetin showed a significant reduction in the levels of proMMP-2, MMP-9, and proMMP-9 in the culture medium. DU-145 cells did not secrete TIMP-1, and the drugs did not alter the secretion of TIMP-2. This work highlights the role played by COX in disturbing the balance between MMPs and TIMPs in prostate cancer cells, and it points to the potential use of COX inibitors, especially COX-2 selective inhibitors, in the prevention and therapy of prostate cancer invasion.
|Elevated levels of circulating plasma matrix metalloproteinase 9 in non-small cell lung cancer patients. |
Iizasa, T, et al.
Clin. Cancer Res., 5: 149-53 (1999) 1999
Elevated expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 have been implicated as playing important roles in tumor invasion and metastasis in various tissues. We investigated the relationship between circulating plasma MMP-9, its expression in tumor samples, and other clinical features in patients with non-small cell lung cancer (NSCLC). A series of 73 patients (45 men and 28 women) who underwent surgery for NSCLC was used in this study. Preoperative plasma concentrations of MMP-9 were examined using a one-step sandwich enzyme immunoassay. Expression levels of MMP-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were measured in 24 tumor samples by immunohistochemistry. The plasma concentration of MMP-9 in NSCLC patients (71.0 +/- 60.2 ng/ml) was significantly elevated compared to that of healthy volunteers (P < 0.0001). MMP-9 concentrations were elevated in 33 of 73 cases (45.2%), compared with a cutoff value of the mean +/- 2 SD in healthy volunteers. There were statistically significant differences in MMP-9 concentration in adenocarcinoma versus squamous cell carcinoma (P = 0.014) and adenocarcinoma versus large cell carcinoma (P = 0.014). Five of 24 patients (20.8%) had positive immunohistochemical MMP staining of the tumor cell cytoplasm, and two cases had positive staining in the surrounding stromal cells. Plasma MMP-9 concentrations were elevated in 45.2% of NSCLC patients; however, this elevation did not seem to correlate with MMP-9 production by cancer and stromal cells. We concluded that the MMP-9 ELISA could be a beneficial adjunct for assessing the tumor burden of NSCLC, especially for types of squamous cell carcinoma and large cell carcinoma.
|Matrix metalloproteinase expression increases after cerebral focal ischemia in rats: inhibition of matrix metalloproteinase-9 reduces infarct size. |
Romanic, A M, et al.
Stroke, 29: 1020-30 (1998) 1998
BACKGROUND AND PURPOSE: Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade the extracellular matrix and are implicated in numerous pathological conditions including atherosclerosis, inflammation, and tumor growth and metastasis. In the brain, the endothelial cell wall, strengthened by tight junctions, defines the blood-brain barrier (BBB). The extracellular matrix molecules constitute the basement membrane underlying the vasculature and play a critical role in maintaining the integrity of the BBB. After focal stroke, there is a breakdown of the BBB with an associated increase in vascular permeability, inflammatory cell influx, and neuronal cell death. The present study was designed to investigate the effects of MMP expression after stroke. METHODS: Focal stroke was produced by permanent middle cerebral artery occlusion (MCAO) in the rat, and MMP protein expression was measured by Western blot and zymogram analysis over a time course ranging from 6 hours to 30 days (n=32). Immunohistochemistry at 1 and 5 days (n=8 and 6, respectively) was also utilized to characterize the expression of several MMPs and related proteins after stroke, including their cellular source. To test the hypothesis that early increased MMP-9 expression is involved in ischemic brain injury, a neutralizing monoclonal antibody directed against MMP-9 was administered intravenously (n=7 per group) 1 hour before MCAO, and infarct size was measured 24 hours later. RESULTS: MMP expression increased progressively over time after stroke. After 12 hours, significant (P<0.05) MMP-9 activity was observed that reached maximum levels by 24 hours (P<0.001), then persisted for 5 days at this level and returned to basal (zero) levels by 15 days. On the basis of morphological criteria, MMP-9 appeared to stain with endothelial cells and neutrophils identified both within and at the periphery of the infarct within 24 hours of focal ischemia. After 5 days, MMP-9 appeared to stain with macrophages present within the infarcted brain. MMP-2 activity was significantly (P<0.001) increased by 24 hours and was maximum after 5 days following MCAO. MMP-2 appeared to stain with macrophages present within the infarcted region. Unlike MMP-9 and MMP-2, tissue inhibitor of metalloproteinase-1 was identified at comparable levels in both control and ischemic tissue after MCAO. MMP-1 and MMP-3 could not be detected in the brain after focal stroke. When an MMP-9-neutralizing monoclonal antibody was administered systemically, animals exhibited significantly reduced infarct size (ie, a 30% reduction compared with non-immune antibody controls; P<0.05). CONCLUSIONS: These results demonstrate that early increased MMP-9 expression in endothelial cells and infiltrating neutrophils is a significant response to cerebral focal ischemia and that selective inhibition of MMP-9 activity can significantly reduce brain injury after stroke.
|Expression of membrane-type matrix metalloproteinase in human gastric carcinomas. |
Nomura, H, et al.
Cancer Res., 55: 3263-6 (1995) 1995
We have examined the implications of membrane-type matrix metalloproteinase (MT-MMP) for activation of the zymogen of MMP-2 (proMMP-2) in human gastric carcinoma. Northern blot analysis demonstrated exclusive expression of MT-MMP in the carcinoma tissues. Immunohistochemically, MT-MMP was colocalized in the carcinoma cells in almost all MMP-2-positive cases (13 of 14 cases). Gelatin zymography of the culture media showed a correlation of the proMMP-2 activation with MT-MMP expression in the carcinoma cells. A microdissection study indicated that proMMP-2 activation is caused only in the carcinoma cell nests that express MT-MMP but not in the normal gastric mucosa. These results are the first demonstration of the MT-MMP-assisted activation of proMMP-2 in the human gastric carcinoma.
|Production of matrix metalloproteinases and tissue inhibitor of metalloproteinases-1 by human brain tumors. |
Nakagawa, T, et al.
J. Neurosurg., 81: 69-77 (1994) 1994
The role of matrix metalloproteinases (MMP's) and their inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1), in human brain tumor invasion was investigated. Gelatinolytic activity was assayed via gelatin zymography, and four MMP's (MMP-1, MMP-2, MMP-3, and MMP-9) and TIMP-1 were immunolocalized in human brain tumors and in normal brain tissues using monoclonal antibodies. The tissue was surgically removed from 44 patients: glioblastoma (five cases), anaplastic astrocytoma (six cases), astrocytoma (four cases), metastatic tumor (six cases), neurinoma (10 cases), meningioma (10 cases), and normal brain tissue (three cases). Glioblastomas, anaplastic astrocytomas, and metastatic tumors showed high gelatinolytic activity and positive immunostaining for MMP's; TIMP-1 was also expressed in these tumors, but some tumor cells were negative for the antibody. Astrocytomas had low gelatinolytic activity and the tumor cells showed no immunoreactivity for MMP's and TIMP-1. Although neurinomas and meningiomas had only moderate proteinase activity and exhibited positive immunoreactivity for MMP-9, intense expression of TIMP-1 was simultaneously observed in these tumor cells. These findings suggest that MMP's play an important role in human brain tumor invasion, probably due to an imbalance between the production of MMP's and TIMP-1 by the tumor cells.
|The expression of invasive behavior of differentiated squamous carcinoma cell line evaluated by an in vitro invasion model. |
Kawahara, E, et al.
Jpn. J. Cancer Res., 84: 409-18 (1993) 1993
In order to elucidate the factors contributory to the expression of invasiveness of oral squamous cell carcinoma, we conducted biochemical and morphological comparisons of well differentiated squamous carcinoma cell line OSC-19 (oral squamous cell carcinoma) and undifferentiated carcinoma cell line KB, both cultured on 3T3 cell-embedded collagen gel (in vitro invasion model). OSC-19 cells invaded 3T3 cell-embedded collagen gel, while KB cells and OSC-19 cells on 3T3 cell-free gel matrix were less invasive. Cultured OSC-19 cells were characterized by lower proliferating activity, lower secretion of laminin and higher secretion of fibronectin than those of KB cells. Although the basement membrane with deposition of laminin and type IV collagen was formed, it was discontinuous at the invasion front. Gelatin zymography and western blotting showed matrix metalloproteinases (MMP), i.e., 72 kDa gelatinase (MMP-2) and 92 kDa gelatinase (MMP-9). Gelatinolytic activity was assayed, and was higher in OSC-19 cells than in KB cells or OSC-19 cells of the 3T3 cell-free model. By immunohistochemical analysis, MMP-2-positive cells were found scattered in both cell lines without any preferential localization, and the positivity for MMP-9 was localized in the invasion front of OSC-19 cells. These results strongly suggest that the invasiveness of squamous cell carcinoma is well correlated with cell-matrix adhesion by fibronectin and with focal elaboration of metalloproteinases, especially MMP-9, which play a major role in degrading the extracellular matrix components.
|A one-step sandwich enzyme immunoassay for human matrix metalloproteinase 2 (72-kDa gelatinase/type IV collagenase) using monoclonal antibodies |
Fujimoto, N. et al.
Clin. Chim. Acta., 221:91-103 (1993) 1993
|Anti-MMP-2, a.a. 468-483 hMMP2, clone 42-5D11 - Data Sheet|