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Introduction: It is generally accepted that metabolic inflammation in the liver is an important driver of disease progression in NASH and associated matrix remodeling/fibrosis. However, the exact molecular inflammatory mechanisms are poorly defined in human studies. Investigation of key pathogenic mechanisms requires the use of pre-clinical models, for instance for time-resolved studies. Such models must reflect molecular disease processes of importance in patients. Herein we characterized inflammation in NASH patients on the molecular level by transcriptomics and investigated whether key human disease pathways can be recapitulated experimentally in Ldlr−/−.Leiden mice, an established pre-clinical model of NASH. Methods: Human molecular inflammatory processes were defined using a publicly available NASH gene expression profiling dataset (GSE48452) allowing the comparison of biopsy-confirmed NASH patients with normal controls. Gene profiling data from high-fat diet (HFD)-fed Ldlr−/−.Leiden mice (GSE109345) were used for assessment of the translational value of these mice. Results: In human NASH livers, we observed regulation of 65 canonical pathways of which the majority was involved in inflammation (32%), lipid metabolism (16%), and extracellular matrix/remodeling (12%). A similar distribution of pathways across these categories, inflammation (36%), lipid metabolism (24%) and extracellular matrix/remodeling (8%) was observed in HFD-fed Ldlr−/−.Leiden mice. Detailed evaluation of these pathways revealed that a substantial proportion (11 out of 13) of human NASH inflammatory pathways was recapitulated in Ldlr−/−.Leiden mice. Furthermore, the activation state of identified master regulators of inflammation (i.e., specific transcription factors, cytokines, and growth factors) in human NASH was largely reflected in Ldlr−/−.Leiden mice, further substantiating its translational value. Conclusion: Human NASH is characterized by upregulation of specific inflammatory processes (e.g., “Fcγ Receptor-mediated Phagocytosis in Macrophages and Monocytes,” “PI3K signaling in B Lymphocytes”) and master regulators (e.g., TNF, CSF2, TGFB1). The majority of these processes and regulators are modulated in the same direction in Ldlr−/−.Leiden mice fed HFD with a human-like macronutrient composition, thus demonstrating that specific experimental conditions recapitulate human disease on the molecular level of disease pathways and upstream/master regulators.

Over the last 4 years, the ERP Organ function on-a-chip evolved from a seed ERP (2015) into a full ERP (in 2016) and is still further growing towards 2018. The program has three use cases: gut-, liver- and lung-function on a chip. In addition nano-detection of biochemical (proteins) was explored.

Organic anion-transporting polypeptide 1B1 (OATP1B1) is an important hepatic uptake transporter, of which the polymorphic variant OATP1B1*15 (Asn130Asp and Val174Ala) has been associated with decreased transport activity. Rosuvastatin is an OATP1B1 substrate and often concomitantly prescribed with oral antidiabetics in the clinic. The aim of this study was to investigate possible drug-drug interactions between these drugs at the level of OATP1B1 and OATP1B1*15. We generated human embryonic kidney (HEK)293 cells stably overexpressing OATP1B1 or OATP1B1*15 that showed similar protein expression levels of OATP1B1 and OATP1B1*15 at the cell membrane as measured by liquid chromatography-tandem mass spectrometry. In HEK-OATP1B1*15 cells, the Vmax for OATP1B1-mediated transport of E217b-G (estradiol 17b-D-glucuronide) was decreased

Objective: Stiffening of the joint is a feature of knee osteoarthritis (OA) that can be caused by fibrosis of the synovium. The infrapatellar fat pad (IPFP) present in the knee joint produces immune-modulatory and angiogenic factors. The goal of the present study was to investigate whether the IPFP can influence fibrotic processes in synovial fibroblasts, and to determine the role of transforming growth factor β (TGFβ) and prostaglandin F2α (PGF2α ) in these processes. Methods: Batches of fat-conditioned medium (FCM) were made by culturing pieces of IPFP obtained from the knees of 13 patients with OA. Human OA fibroblast-like synoviocytes (FLS) (from passage 3) were cultured in FCM with or without inhibitors of TGFβ/activin receptor-like kinase 5 or PGF2α for 4 days. The FLS were analyzed for production of collagen and expression of the gene for procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2; encoding lysyl hydroxylase 2b, an enzyme involved in collagen crosslinking) as well as the genes encoding α-smooth muscle actin and type I collagen α1 chain. In parallel, proliferation and migration of the synoviocytes were analyzed. Results: Collagen production and PLOD2 gene expression by the FLS were increased 1.8-fold (P < 0.05) and 6.0-fold (P < 0.01), respectively, in the presence of FCM, relative to control cultures without FCM. Moreover, the migration and proliferation of synoviocytes were stimulated by FCM. Collagen production was positively associated with PGF2α levels in the FCM (R = 0.89, P < 0.05), and inhibition of PGF2α levels reduced the extent of FCM-induced collagen production and PLOD2 expression. Inhibition of TGFβ signaling had no effect on the profibrotic changes. Conclusion: These results indicate that the IPFP can contribute to the development of synovial fibrosis in the knee joint by increasing collagen production, PLOD2 expression, cell proliferation, and cell migration. In addition, whereas the findings showed that TGFβ is not involved, the more recently discovered profibrotic factor PGF2α appears to be partially involved in the regulation of profibrotic changes.

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Matrix metalloproteinases (MMPs) may play a pathophysiological role in the development of diabetic nephropathy (DN). We hypothesized that urinary MMP activity in patients with type 2 diabetes mellitus (T2DM) is related to a decline in renal function. We determined MMP-2, -8 and -9 activity in 24-h urine collections in relation to risk factors for DN in T2DM patients with (UA, n=27) and without albuminuria (NA, n=48) and controls (CO, n=28). MMP-8 and -9 levels were highest in UA patients (P

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Introduction. Matrix metalloproteinase 9 (MMP-9) is an endopeptidase involved in various cellular processes, such as tumour development and metastatic spread. In biological samples, MMP-9 can occur as pro-MMP-9 and active MMP-9, or these factors complexed with the inhibitor TIMP-1. An assay, which can measure active and total MMP-9 in biological samples, has been used on the urine from bladder cancer patients and demonstrated a significant correlation between MMP-9 and clinical parameters. The prognostic value of these measurements has never been investigated. Using this assay we have investigated the prognostic influence of total and active MMP-9 in urine from bladder cancer patients. Material and methods. Fresh voided urines from 188 consecutive patients diagnosed with bladder cancer were collected and frozen at diagnosis. After 15 years follow-up 13 patients were still alive, and 175 patients had died. MMP-9 was measured with an immunocapture activity assay. Results. Median MMP-9total was 173.7 units/10 g creatinine (range 034 792), and median MMP-9active was 14 units/10g creatinine (range, 0294 757). The two factors were correlated (Spearmańs rho 0.74, p

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Background: Matrix metalloproteinase-9 (MMP-9) is thought to play a central role in abdominal aortic aneurysm (AAA) initiation. Doxycycline, a tetracycline analogue, has direct MMP-9-inhibiting properties in vitro, and it effectively suppresses AAA development in rodents. Observed inhibition of AAA progression, and contradictory findings in human studies evaluating the effect of doxycycline therapy on aortic wall MMP-9, suggest that the effects of doxycycline extend beyond MMP-9 inhibition and that the effect may be dose-dependent. Methods: This clinical trial evaluated the effect of 2 weeks of low- (50 mg/d), medium- (100 mg/d), or high-dose (300 mg/d) doxycycline vs no medication in four groups of 15 patients undergoing elective AAA repair. The effect of doxycycline treatment on MMP and cysteine proteases, and their respective inhibitors, was evaluated by quantitative polymerase chain reaction, Western blot analysis, immunocapture protease activity assays, and immunohistochemistry. Results: Doxycycline was well tolerated and no participants dropped out. Doxycycline treatment reduced aortic wall MMP-3 and MMP-25 messenger RNA expression (P < .045 and P < .014, respectively), selectively suppressed neutrophil collagenase and gelatinase (MMP-8 and MMP-9) protein levels (P < .013 and

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Background: Infarct rupture is a usually fatal complication of myocardial infarction (MI), for which no molecular mechanism has been described in humans. Experimental evidence in mouse models suggests that the degradation of the extracellular matrix by matrix metalloproteinases (MMPs) plays an important role in infarct rupture. The present study was designed to study the role of MMP-2, MMP-8, and MMP-9 in human infarct rupture. Methods: Heart samples were obtained from patients who died from infarct rupture and control MI patients. The MMP activity was determined by zymography and quantitative immunocapture activity assay. TIMP-1 levels were measured and immunohistochemistry for MMP-2 and MMP-9 was performed. Results: The amounts of both total and active MMP-8 and MMP-9 were significantly higher in ruptured infarct tissue than in control MI tissue, but no differences in MMP-2 activity were observed. Furthermore, the number of inflammatory cells was significantly higher in the ruptured infarcts than in control infarcts. Conclusions: These data suggest that increased MMP-8 and MMP-9 activity in the infarct area, caused by a more prominent infiltration of inflammatory cells, contribute to infarct rupture in humans. © 2009 Elsevier Inc. All rights reserved.

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Over-expression of matrilysin (MMP-7) is predominantly associated with epithelial (pre)malignant cells. In the present study MMP-7 expression is also found in endothelial cells in various human cancer types. Endothelial MMP-7 was associated with CD34 and/or CD105 expression. These immunohistochemical data were confirmed by RT-PCR on VEGF-stimulated endothelial cells. In addition, MMP-7 was also identified in sprouting endothelial cells in vitro. The potential clinical relevance of endothelial MMP-7 was assessed for cervical cancer patients by evaluating the association with overall survival. In contrast to MMP-7 in malignant epithelial cells, MMP-7 expression in endothelial cells showed a significant association with poor survival (LR 5.12, P = 0.02, n = 30). Our data suggest that MMP-7 is involved in tumor angiogenesis, thereby contributing to malignant growth and hence associated with decreased survival. © 2007 International Society of Matrix Biology.

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Objective: To analyse the effects of leflunomide and methotrexate treatment on matrix metalloproteinase (MMP) activity levels in a2 macroglobulin/MMP (α2M/MMP) complexes in the systemic circulation of rheumatoid arthritis (RA) patients. Methods: A total of 102 RA patients from a prospective, double-blind, randomised clinical trial comparing leflunomide and methotrexate were selected; clinical data and blood samples were collected at baseline, at 4 months and at 1 year. Serum MMP activity levels in α2M were quantified using low molecular weight fluorogenic substrates, indicating the proportion of activated MMPs that were not inhibited by specific tissue inhibitors of MMP (TIMP). Results: Patients had active disease as shown by high disease activity score (DAS, mean of 6.9 and 7.0 for methotrexate and leflunomide patients respectively), which was reduced over the study period (4.2 and 5.2 respectively, p

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Growth and rupture of abdominal aortic aneurysms (AAAs) result from increased collagen turnover. Collagen turnover critically depends on specific collagenases that cleave the triple helical region of fibrillar collagen. As yet, the collagenases responsible for collagen degradation in AAAs have not been identified. Increased type I collagen degradation products confirmed collagen turnover in AAAs (median values: 80%). We found indications that this secondary deficiency relates to cystatin C degradation by (neutrophil-derived) proteases. Copyright © American Society for Investigative Pathology.

Renal expression of MMP-2, -9, and tissue inhibitor of MMP-1 (TIMP-1) correlates with histological disease activity in anti-neutrophil cytoplasm autoantibody (ANCA)-associated vasculitis (AAV). We studied whether urinary and plasma levels of MMP-2, -9, and TIMP-1 reflect renal expression of these proteins and renal disease-activity in AAV. Urine and plasma samples of patients with AAV who underwent a renal biopsy were collected (n = 32). Urinary activity of MMP-2 and -9 was measured by activity assays. Urinary and plasma levels of MMP-2, MMP-9, and TIMP-1 proteins were measured by ELISA. Healthy controls provided plasma and urine for comparison (n = 31). In patients, the relationship of urinary and plasma levels with renal expression of MMP-2 and MMP-9 and clinical and histological disease activity was studied. Renal MMP expression was compared between patients and controls (n = 8). Urinary MMP-2 and MMP-9 activity and urinary and plasma TIMP-1 levels were significantly higher in patients than in controls. In glomeruli of patients, both MMP-2 and MMP-9 expression reflected active glomerular inflammation. Urinary activity of MMP-2 and MMP-9 did not correlate with renal MMP expression or plasma levels. Urinary MMP activity correlated negatively with glomerular inflammation, but positively with fibrous crescents. Urinary MMP-2 and TIMP-1 levels showed a positive correlation with tubulointerstitial damage and a negative correlation with creatinine clearance. Urinary MMP-2, MMP-9, and TIMP-1 are elevated in AAV but do not reflect renal MMP expression and glomerular inflammation. However, urinary MMP-2 activity and TIMP-1 levels reflect tubulointerstitial damage and correlate negatively with creatinine clearance at biopsy. Copyright © 2007 the American Physiological Society.

The scavenger receptor CD163 is selectively expressed on tissue macrophages and human monocytes. CD163 has been implicated to play a role in the clearance of hemoglobin and in the regulation of cytokine production by macrophages. Membrane CD163 can be cleaved by matrix metalloproteinases (MMP) resulting in soluble CD163 (sCD163). In the present report the shedding of CD163 was investigated in multiple sclerosis (MS). An upregulation of plasma sCD163 and a down regulation of membrane CD163 in MS patients compared to healthy controls was observed. The levels of plasma sCD163 correlated with plasma MMP-9 levels in controls, but not in MS patients. Moreover, evidence was obtained for CD163-cleaving MMP activity in plasma of MS patients. Finally, the increased proteolytic shedding of CD163 correlated to reduced plasma levels of circulating inflammatory cytokines. Collectively, our results provide evidence for proteolytic shedding of CD163 in MS and suggest a possible link to cytokine production. © 2007 Elsevier B.V. All rights reserved.

Recently, complexes of matrix metalloproteinase matrix metalloproteinase-9 (MMP-9) with lipocalin-2 (neutrophil gelatinase-associated lipocalin) were found in the urine obtained from breast cancer patients, while these were completely absent in that obtained from healthy controls. In vitro data suggested a possible role for lipocalin-2 in the protection of MMP-9 against autolysis. To establish this effect in vivo, we determined the presence of MMP-9, lipocalin-2 and their complex in tumour tissue from 81 gastric cancer patients. The effect of the presence of the individual parameters, the complexes, and the inhibitors TIMP-1 and TIMP-2 on MMP-9 activity was evaluated with a bioactivity assay. Immuno-histochemical (double) staining identified epithelial cells as the most likely cellular source. Finally, evaluation of all these parameters with clinico-pathological scores revealed that tumour MMP-9/lipocalin-2 complexes were significantly related with the classifications of Laurén and WHO, and highly associated with worse survival in Cox's univariate (HR 2.087, P = 0.006) and multivariate analyses (HR 2.095, P = 0.025). © 2007 Elsevier Ltd. All rights reserved.

OBJECTIVE - Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a multi-system disorder characterized by progressive cyst formation in the kidneys. Serious complications of ADPKD are intracranial and aortic aneurysms. The condition is mainly caused by mutations in the PKD1 or PKD2 gene. We have carefully analyzed vascular remodeling in hypomorphic Pkd1 mouse model with dissecting aneurysms in the aorta. METHODS AND RESULTS - Quantitative real-time polymerase chain reaction revealed that in the aorta the expression of normal Pkd1 is reduced to approximately 26%. Using (immuno)histochemistry we have characterized the pathogenetic sequence for dissecting aneurysm formation. The aorta shows regions with accumulation of matrix components between the elastin lamellae. This is followed by increased numbers of smooth muscle cells and locally weakening of the media. In the intima, accumulation of matrix components and detachment of endothelial cells from the elastin lamellae results in a tear. The combination of weak media and a tear in the intima leads to rupture of the vessel wall resulting in intramural bleeding. CONCLUSIONS - The Pkd1 mouse reveals that polycystin1 is implicated in maintenance of the vessel wall structural integrity, and it is a useful model for dissecting aneurysm formation studies. © 2007 American Heart Association, Inc. Chemicals / CAS: calcium, 7440-70-2; collagen, 9007-34-5; elastin, 9007-58-3; fibronectin, 86088-83-7; gelatinase A, 146480-35-5; gelatinase B, 146480-36-6; osteopontin, 106441-73-0; perlecan, 143972-95-6; versican, 126968-45-4; polycystic kidney disease 1 protein; RNA, Messenger; TRPP Cation Channels

Background: Previous studies have shown an upregulation of matrix metalloproteinases (MMPs) in intestinal tissue of patients with inflammatory bowel disease (IBD) and significant clinical improvement after administration of the anti-TNF-α antibody infliximab. The aims of our study were to determine expression and secretion of MMP-1, -2, -3, -9, and their inhibitors TIMP-1, -2 by IBD versus control intestinal mucosa ex vivo and to assess the regulatory capacity by infliximab of the proteolytic phenotype. Methods: Intestinal mucosal explants from 20 IBD and 15 control patients were cultured with or without infliximab and/or the T-cell activator pokeweed mitogen (PWM). Explants and culture supernatants were analyzed for MMPs, TIMPs, and TNF-α protein, activity and/or mRNA levels. All patients were genotyped for functional TNF-α, MMP, and TIMP single nucleotide polymorphism (SNP) loci. Results: Expression of MMP and TIMP protein/activity in basal medium was higher in IBD versus control explants. Dependent on genotype at SNP loci, infliximab downregulated MMP-1, -3, and -9 relative to TIMP-1 and -2 and also decreased MMP-1 and -3 activities, while PWM enhanced these levels, partly counteracted again by infliximab. The expression of MMP-2 relative to TIMP did not change by treatment with infliximab and/or PWM. Conclusions: The high expression of MMPs in patients with IBD suggests a role for these proteinases in the pathogenesis of this disease. Infliximab seems to induce a genotype-associated matrix protective phenotype, which may contribute to the observed therapeutic efficacy of this drug in IBD, particularly at the mucosal surface. Copyright © 2006 Crohn's & Colitis Foundation of America, Inc.

Healthy bone is a rigid yet living tissue that undergoes continuous remodeling. Osteoclasts resorb bone in the remodeling cycle. They secrete H+-ions and proteinases to dissolve bone mineral and degrade organic bone matrix, respectively. One of the main collagenolytic proteinase in osteoclasts is cathepsin K, a member of papain family cysteine proteinases. Recently, it has been shown that osteoblasts may contribute to organic matrix remodeling. We therefore investigated their ability to produce cathepsin K for this action. Trabecular bone samples were collected from patients operated due to a fracture of the femoral neck. Part of the bone was decalcified and the rest was used for cell isolation. Sections from the decalcified bone were immunostained with antibodies against cathepsin K. Isolated cells were characterized for their ability to form mineralized matrix and subsequently analyzed for their cathepsin K production by Western blotting and quantitative RT-PCR. Osteoblasts, bone lining cells and some osteocytes in situ showed cathepsin K immunoreactivity and osteoblast-like cells in vitro produced cathepsin K mRNA and released both 42 kDa pro- and 27 kDa processed cathepsin K to culture media. Osteoblastic cathepsin K may thus contribute to collagenous matrix maintenance and recycling of improperly processed collagen I. Whether osteoblastic cathepsin K synthesis has consequences in diseases characterized by abnormal bone matrix turnover remains to be investigated. © 2006. Chemicals / CAS: cathepsin K, 94716-09-3; Alkaline Phosphatase, EC 3.1.3.1; cathepsin K, EC 3.4.22.-; Cathepsins, EC 3.4.-; Core Binding Factor Alpha 1 Subunit; Matrix Metalloproteinase 13, EC 3.4.24.-; Osteoprotegerin; RANK Ligand; RNA, Messenger; RUNX2 protein, human; TNFSF11 protein, human; Tumor Necrosis Factor-alpha

Background and Purpose - We studied matrix metalloproteinases (MMP) 2, 8, and 9 and extracellular matrix metalloproteinase inducer (EMMPRIN) levels in relation to carotid atherosclerotic plaque characteristics. Methods - Carotid atherosclerotic plaques (n=150) were stained and analyzed for the presence of collagen, smooth muscle cell (SMC), and macrophages. Adjacent segments were used to isolate total protein to assess MMP-2 and MMP-9 activities and gelatin breakdown, MMP-8 activity, and EMMPRIN levels. Results - Macrophage-rich lesions revealed higher MMP-8 and MMP-9 activities, whereas SMC-rich lesions showed higher MMP-2 activity. The levels of less glycosylated EMMPRIN-45kD were higher in SMC-rich lesions and lower in macrophage-rich plaques. EMMPRIN-45kD was associated with MMP-2 levels, whereas EMMPRIN-58kD was related to MMP-9 levels. Conclusions - MMP-2, MMP-8, and MMP-9 activities differed among carotid plaque phenotypes. Different EMMPRIN glycosylation forms are associated with either MMP-2 or MMP-9 activity, which suggests that EMMPRIN glycosylation may play a role in MMP regulation and plaque destabilization. © 2005 American Heart Association, Inc. Chemicals / CAS: collagen, 9007-34-5; gelatin, 9000-70-8; gelatinase A, 146480-35-5; gelatinase B, 146480-36-6; Antigens, CD147, 136894-56-9; Collagen, 9007-34-5; Matrix Metalloproteinase 2, EC 3.4.24.24; Matrix Metalloproteinase 8, EC 3.4.24.34; Matrix Metalloproteinase 9, EC 3.4.24.35

The hallmark of fibrotic processes is an excessive accumulation of collagen. The deposited collagen shows an increase in pyridinoline cross-links, which are derived from hydroxylated lysine residues within the telopeptides. This change in cross-linking is related to irreversible accumulation of collagen in fibrotic tissues. The increase in pyridinoline cross-links is likely to be the result of increased activity of the enzyme responsible for the hydroxylation of the telopeptides (telopeptide lysyl hydroxylase, or TLH). Although the existence of TLH has been postulated, the gene encoding TLH has not been identified. By analyzing the genetic defect of Bruck syndrome, which is characterized by a pyridinoline deficiency in bone collagen, we found two missense mutations in exon 17 of PLOD2, thereby identifying PLOD2 as a putative TLH gene. Subsequently, we investigated fibroblasts derived from fibrotic skin of systemic sclerosis (SSc) patients and found that PLOD2 mRNA is highly increased indeed. Furthermore, increased pyridinoline cross-link levels were found in the matrix deposited by SSc fibroblasts, demonstrating a clear link between mRNA levels of the putative TLH gene (PLOD2) and the hydroxylation of lysine residues within the telopeptides. These data underscore the significance of PLOD2 in fibrotic processes. Chemicals/CAS: collagen, 9007-34-5; procollagen lysine 2 oxoglutarate 5 dioxygenase, 9059-25-0; Collagen, 9007-34-5; Cross-Linking Reagents; DNA, Complementary; Peptides; PLOD2 protein, human, EC 1.14.11.4; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase, EC 1.14.11.4; RNA, Messenger

Background. Bisphosphonates reduce the bone metastasis formation and angiogenesis but the exact molecular mechanisms involved are unclear. Progelatinase A (proMMP-2; 78 KDa) is activated up during the tumor spread and metastasis by a cell surface-associated matrix metalloproteinase (membrane-type matrix metalloproteinase [MT1-MMP] or MMP-14). Material and methods. We evaluated the effects of a bisphosphonate (clodronate) on MT1-MMP mRNA expression and protein production, catalytic activity and proteolytic activation of proMMP-2 by cultured human MG-63 osteosarcoma cells. Results. Clodronate, at therapeutically attainable noncytotoxic concentrations, dose-dependently inhibited phorbol myristic acetate (PMA)-induced proteolytic activation of proMMP-2 by human MG-63 osteosarcoma cells. Clodronate also downregulated the PMA-induced expression of MT1-MMP mRNA and protein production in human MG-63 osteosarcoma cells, as evidenced by Northern analysis and fluorescent immunohistochemistry. Furthermore, clodronate inhibited directly and dose-dependently MT1-MMP activity, and the MT1-MMP inhibition by clodronate was reduced in the presence of an increased (5 mM) Ca2+ concentrations when compared to physiological (1 mM) Ca2+ concentrations. Conclusion. We conclude that (1) the extracellular/cell-associated mechanism of bisphosphonate involves inhibition of MT1-MMP catalytic activity eventually by chelation, and that (2) intracellular mechanism involves downregulation of induced MT1-MMP mRNA and protein expression. The inhibition and downregulation of MT1-MMP by clodronate can be related to their ability to reduce MG-63 osteosarcoma cell invasion and spread. These findings may, at least in part, explain at molecular level the antitumor and antibone resorption activities of clodronate observed in clinical studies. © 2003 Elsevier Inc. All rights reserved.

Purpose: On the basis of the finding of marked overexpression in angiogenic microvessels, aminopeptidase N/CD13 has recently been suggested to play a prominent role in tumor angiogenesis. A soluble form of CD13 (sCD13) is present in human plasma, but its role in cancer has not been addressed. We hypothesized that sCD13 would be shed by tumor cells and/or endothelial cells lining tumor vessels, giving high levels of sCD13 in intratumoral fluid (TF) deposits and in malignant effusions. If so, sCD13 could be a convenient potential marker for tumor load and/or activated tumor endothelium. Experimental Design: We have measured the specific sCD13 activity in effusions from 90 cancer patients and 12 patients with a nonmalignant condition, and studied its relationship with other major (anti-) angiogenic factors. In a separate group of patients (n = 41), the relationship of sCD13 activity in plasma with tumor load was studied. Results: The sCD13 activity was highest in plasma from cancer patients 71.9 (fmol/ml/s hydrolyzed substrate) versus 42.4 for healthy subjects. In TF, malignant effusions, and nonmalignant effusions, the activities were 52.8, 33.5, and 18.6, respectively. We further studied the relationship of sCD13 with tumor load as well as with vascular endothelial growth factor (VEGF), endostatin, matrix metalloproteinase (MMP)-2, MMP-9, urokinase-type plasminogen activator, and plasmin. A significant correlation of sCD13 activity in plasma was found with tumor load (r = 0.68; P = 0.01), suggesting that plasma sCD13 is, at least, partly originating from tumor(-endothelium). The concentrations of VEGF and endostatin and the activities of urokinase-type plasminogen activator and MMP-9, but not MMP-2, were significantly higher in TF compared with all other effusions. In TF, a correlation between sCD13 and VEGF was found (r = 0.67; P = 0.03). No correlation of sCD13 with the other protease activities was found. Conclusion: The sCD13 activity is elevated in plasma and effusions of cancer patients. A strong correlation of plasma sCD13 with tumor load was found. On the basis of these results, the potential of sCD13 activity as a tumor and/or angiogenesis marker warrants further investigation. Chemicals/CAS: Antigens, CD13, EC 3.4.11.2; Collagen, 9007-34-5; Endostatins; Endothelial Growth Factors; Fibroblast Growth Factor 2, 103107-01-3; Intercellular Signaling Peptides and Proteins; Lymphokines; Matrix Metalloproteinase 2, EC 3.4.24.24; Matrix Metalloproteinase 9, EC 3.4.24.35; Peptide Fragments; Plasmin, EC 3.4.21.7; Tumor Markers, Biological; Urinary Plasminogen Activator, EC 3.4.21.73; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Matrix metalloproteinases (MMP) types 2 and 9 (also known as gelatinase A and B) are thought to be causally involved in cancer invasion and metastasis. In normal as well as in malignant tissue, both these MMPs occur in multiple forms such as inactive precursors, active enzymes and enzyme- inhibitor complexes. Using newly developed quantitative activity assays, the levels of active MMP-2, total (active and activatable) MMP-2 and total MMP-9 were found to be significantly higher in breast carcinomas than in fibroadenomas. In addition, active MMP-2 and MMP-9 were detected more frequently in malignant than in benign breast carcinoma. These new quantitative activity assays are likely to be of use in studying the mechanism of action of both MMP-2 and -9, assessing their potential prognostic value in different cancers and in the design of MMP inhibitors for preventing cancer metastasis. (C) 2000 Wiley-Liss, Inc. Chemicals/CAS: Matrix Metalloproteinase 2, EC 3.4.24.24; Matrix Metalloproteinase 9, EC 3.4.24.35

Gelatinase-B has been shown to be produced by a variety of different cells, fibroblasts in particular. It has also been shown to be elevated in the sera of patients with specific disease states, for example hepatocellular carcinoma. A novel technology is described for determining the activity of matrix metalloproteinases (MMPs) using a colorimetric assay. The assay is based on a modified pro-urokinase constructed by protein engineering. In the modified prourokinase the activation sequence, normally recognised by plasmin (Pro-Arg-Phe-Lys # Ile-Ile-Gly-Gly), was replaced by a sequence that is specifically recognised by MMPs (Arg-Pro-Leu-Gly # Ile-Ile-Gly-Gly). A chromogenic peptide substrate for urokinase is then used to measure the active urokinase, generated through MMP activation of the modified urokinase. The assay uses a MMP-9 specific monoclonal antibody, coated 96 well microtitre plate to confer MMP-9 specificity. MMP-9 is captured from biological fluids or cell culture media through overnight incubation of the samples at 4°C, in the microtitre plate wells. Immobilised MMPs are then activated using APMA for a 2hour incubation period at 37°C. Differential activation with APMA, enables the analysis of the activity of both the active and latent forms of MMP-9. Detection incubation with modified urokinase and the substrate S-2444 continues at 37°C for 2 to 6 hours. The normal assay range is between 32 and 2ng/ml for a 2 hour incubation. A more sensitive assay range between 8 and 0.5ng/ml, enabled a detection sensitivity of 0.5ng/ml for a 6 hour detection incubation period. Cell culture and serum/plasma samples were used to validate the activity assay. U937 cell cultures, stimulated under different conditions, were assayed in the activity assay and in a variety of MMP-9 specific ELISAs. The APB RPN 2614 ELISA used, specifically detects pro MMP-9. In addition other antibodies were used to construct ELISAs for the measurement of total MMP-9 and pro and active MP-9/TIMP- 1 complexes. Further validation work was performed using serum and a variety of plasma types, for linearity and recovery experiments. It was observed that greater than a 1:16 dilution of these samples was required to obtain a high recovery. Normal latent MMP-9 levels were found to be detectable at this dilution. MMP-9 activity in normal biological samples was detected using the novel activity assay. The activity assay results were shown to correlate with results obtained from the MMP-9 specific ELIS As. A separate study determined the levels of Gelatinase B (MMP-9) activity in saliva samples from patients with Sjögren s syndrome. An assay based on radiolabelled gelatinated collagen showed that gelatinase activity was slightly, although not significantly, increased in Sjögren s syndrome. The novel immunocapture activity assay however, specifically measuring MMP-9, showed that MMP-9 levels were increased in Sjögren s patients compared with healthy controls: MMP-9 (already active): patients 8.9 + 2.5 U/mg, controls 1.0+0.5 U/mg (p=0.002); latent plus active MMP-9: patients 53.1 ±9.8 U/mg, controls 16.5 + 2.6 U/mg (p=0.010). © Warcourt Brace & Co. Ltd 1999.