Elevated expression levels of lysyl oxidases protect against aortic aneurysm progression in Marfan syndrome.

Patients with Marfan syndrome (MFS) are at high risk of life-threatening aortic dissections. The condition is caused by mutations in the gene encoding fibrillin-1, an essential component in the formation of elastic fibers. While experimental findings in animal models of the disease have shown the involvement of transforming growth factor-ß (TGF-ß)- and angiotensin II-dependent pathways, alterations in the vascular extracellular matrix (ECM) may also play a role in the onset and progression of the aortic disease. Lysyl oxidases (LOX) are extracellular enzymes, which initiates the formation of covalent cross-linking of collagens and elastin, thereby contributing to the maturation of the ECM. Here we have explored the role of LOX in the formation of aortic aneurysms in MFS. We show that aortic tissue from MFS patients and MFS mouse model (Fbn1(C1039G/+)) displayed enhanced expression of the members of the LOX family, LOX and LOX-like 1 (LOXL1), and this is associated with the formation of mature collagen fibers. Administration of a LOX inhibitor for 8weeks blocked collagen accumulation and aggravated elastic fiber impairment, and these effects correlated with the induction of a strong and rapidly progressing aortic dilatation, and with premature death in the more severe MFS mouse model, Fbn1(mgR/mgR), without any significant effect on wild type animals. This detrimental effect occurred preferentially in the ascending portion of the aorta, with little or no involvement of the aortic root, and was associated to an overactivation of both canonical and non-canonical TGF-ß signaling pathways. The blockade of angiotensin II type I receptor with losartan restored TGF-ß signaling activation, normalized elastic fiber impairment and prevented the aortic dilatation induced by LOX inhibition in Fbn1(C1039G/+) mice. Our data indicate that LOX enzymes and LOX-mediated collagen accumulation play a critical protective role in aneurysm formation in MFS.

Drug-drug interactions between rosuvastatin and oral antidiabetic drugs occurring at the level of OATP1B1.

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 V(max) for OATP1B1-mediated transport of E(2)17ß-G (estradiol 17ß-d-glucuronide) was decreased >60%, whereas K(m) values (Michaelis constant) were comparable. Uptake of rosuvastatin in HEK-OATP1B1 cells (K(m) 13.1 ± 0.43 µM) was nearly absent in HEK-OATP1B1*15 cells. Interestingly, several oral antidiabetics (glyburide, glimepiride, troglitazone, pioglitazone, glipizide, gliclazide, and tolbutamide), but not metformin, were identified as significant inhibitors of the OATP1B1-mediated transport of rosuvastatin. The IC(50) values for inhibition of E(2)17ß-G uptake were similar between OATP1B1 and OATP1B1*15. In conclusion, these studies indicate that several oral antidiabetic drugs affect the OATP1B1-mediated uptake of rosuvastatin in vitro. The next step will be to translate these data to the clinical situation, as it remains to be established whether the studied oral antidiabetics indeed affect the clinical pharmacokinetic profile of rosuvastatin in patients.

Cooperative effect of TNFalpha, bFGF, and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix. Role of urokinase activity.

In angiogenesis associated with tissue repair and disease, fibrin and inflammatory mediators are often involved. We have used three-dimensional fibrin matrices to investigate the humoral requirements of human microvascular endothelial cells (hMVEC) to form capillary-like tubular structures. bFGF and VEGF165 were unable to induce tubular structures by themselves. Simultaneous addition of one or both of these factors with TNFalpha induced outgrowth of tubules, the effect being the strongest when bFGF, VEGF165, and TNFalpha were added simultaneously. Exogenously added u-PA, but not its nonproteolytic amino-terminal fragment, could replace TNFalpha, suggesting that TNFalpha-induced u-PA synthesis was involved. Soluble u-PA receptor (u-PAR) or antibodies that inhibited u-PA activity prevented the formation of tubular structures by 59-99%. epsilon-ACA and trasylol which inhibit the formation and activity of plasmin reduced the extent of tube formation by 71-95%. TNFalpha or u-PA did not induce tubular structures without additional growth factors. bFGF and VEGF165 enhanced of the u-PAR by 72 and 46%, but TNFalpha itself also increased u-PAR in hMVEC by 30%. Induction of mitogenesis was not the major contribution of bFGF and VEGF165 because the cell number did not change significantly in the presence of TNFalpha, and tyrphostin A47, which inhibited mitosis completely, reduced the formation of tubular structures only by 28-36%. These data show that induction of cell-bound u-PA activity by the cytokine TNFalpha is required in addition to the angiogenic factors VEGF165 and/or bFGF to induce in vitro formation of capillary-like structures by hMVEC in fibrin matrices. These data may provide insight in the mechanism of angiogenesis as occurs in pathological conditions.

Leflunomide and methotrexate reduce levels of activated matrix metalloproteinases in complexes with alpha2 macroglobulin in serum of rheumatoid arthritis patients.

OBJECTIVE: To analyse the effects of leflunomide and methotrexate treatment on matrix metalloproteinase (MMP) activity levels in alpha2 macroglobulin/MMP (alpha2M/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 alpha2M 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<0.001). In leflunomide-treated patients a significant reduction of MMP activity levels was observed as early as at the 4 months timepoint persisting thereafter, whereas in methotrexate-treated patients the reduction was seen at 1 year. CONCLUSION: The results show that systemic levels of activated MMPs are reduced in RA patients upon exposure to leflunomide or methotrexate.

Gingival tissue and crevicular fluid co-operation in adult periodontitis.

Activated matrix metalloproteinase-3 (MMP-3) can contribute to periodontal ligament destruction in adult periodontitis. Since MMP-3 has been reported to activate proMMP-8 and -9, it was speculated that gingival tissue fibroblast-derived MMP-3 might, in periodontitis, be responsible for activation of gingival crevicular fluid (GCF) neutrophil-derived proMMP-8 and -9. Immunohistochemistry disclosed MMP-3 in gingival fibroblasts in periodontitis. Cultured gingival fibroblasts released only pro-MMP-3 when stimulated with tumor necrosis factor-alpha. However, Western blot revealed partially activated MMP-3, MMP-8, and MMP-9 in periodontitis GCF. Active MMP-8 (p < 0.05) and MMP-9 (p < 0.05) correlated with the presence of active MMP-3. It seems that resident gingival fibroblasts produce pro-MMP-3 in GCF, where it becomes activated, probably by cathepsin G or elastase released by neutrophils. Active MMP-3 then activates neutrophil-derived pro-MMP-8 and -9. Different tissue compartments/cells exert co-operative actions in mutual local MMP activation cascades.

Metalloproteinase inhibition reduces constrictive arterial remodeling after balloon angioplasty: a study in the atherosclerotic Yucatan micropig.

BACKGROUND: Arterial remodeling after balloon angioplasty has been recognized as a major determinant of restenosis. Perturbation of collagen metabolism might be important. After balloon injury, matrix metalloproteinase (MMP) expression is upregulated. We investigated the effect of Batimastat, a nonspecific MMP inhibitor, on late lumen loss, arterial remodeling, and neointima formation after balloon dilation. METHODS AND RESULTS: In atherosclerotic iliac arteries of 12 Yucatan micropigs, balloon dilation was performed, with intravascular ultrasound and quantitative angiography used before and after balloon dilation and at 42-day follow-up. The animals were randomly divided into 2 groups, the Batimastat group (n=6) and the vehicle group (n=6). All animals were intraperitoneally injected with either Batimastat or a vehicle immediately after balloon dilation and at 2 weeks and 4 weeks after balloon dilation. Angiographic and echographic late lumen loss in the Batimastat group versus the vehicle group was 0.3+/-0.1 versus 0.8+/-0.1 mm (P=0.01) and 2.2+/-0.5 versus 4.9+/-0.7 mm(2) (P=0.004), respectively. Late media-bounded area loss was used as a measure of remodeling after balloon dilation and was 0.9+/-0.6 mm(2) in the Batimastat group compared with 3.8+/-0.8 mm(2) in the vehicle group (P=0.003, mixed model analysis P=0.01). Neointima formation was 1.3+/-0.3 mm(2) in the Batimastat group and 1.0+/-0.2 mm(2) in the vehicle group (P=0. 542). CONCLUSIONS: Metalloproteinase inhibition by Batimastat significantly reduced late lumen loss after balloon angioplasty by inhibition of constrictive arterial remodeling, whereas neointima formation was not inhibited by MMP inhibition.

Enhanced urinary gelatinase activities (matrix metalloproteinases 2 and 9) are associated with early-stage bladder carcinoma: a comparison with clinically used tumor markers.

Matrix metalloproteinases (MMPs) are involved in tumor growth and metastasis, promoting the migration and invasion of cells. In this study, the amount of MMP-2 and MMP-9 activity was measured in urine from superficial bladder carcinoma patients (pTa, pT1) to evaluate their possible diagnostic value. The active and total amount of MMP-2 and MMP-9, respectively, in urine from tumor patients were compared with the levels in urine from age- and gender-matched healthy volunteers. Both MMP-2 and MMP-9 activity levels were significantly enhanced in urine from patients with high invasive cancers (pT2, PT3), whereas in urine from healthy controls no or very low MMP activities were found. More importantly, a substantial number of urine samples from patients with superficial tumors contained elevated MMP-2 and MMP-9 activities, suggesting that enhanced urinary MMP activity levels, indeed, might be indicative for early-stage bladder cancer. Overall, urinary MMP-2 and MMP-9 activity levels were significantly correlated to each other, with some individual exceptions. A comparison between urinary MMP-9 activity and a recently proposed urinary marker for bladder cancer, NMP-22, showed slightly lower numbers of patients with elevated levels for MMP-9. But because MMP-9 and NMP-22 levels were not correlated, enhanced urinary MMP activity might be useful as a marker for superficial bladder carcinoma like, or especially in combination with, other markers.

A novel and simple immunocapture assay for determination of gelatinase-B (MMP-9) activities in biological fluids: saliva from patients with Sjögren's syndrome contain increased latent and active gelatinase-B levels.

Here we describe a new principle for accessing the activity of the different members of the human matrix-metalloproteinases (MMPs) by a colorimetric assay. Using protein engineering, a modified pro-urokinase was made in which the activation sequence, normally recognized by plasmin (ProArgPheLys/IleIleGlyGly), was replaced by a sequence that is specifically recognized by MMPs (ArgProLeuGly/IleIleGlyGly). The active urokinase resulting from the activation of this modified pro-urokinase by MMPs can be measured directly using a chromogenic peptide substrate for urokinase. The assay has been made specific for MMP-9 using an MMP-9 specific monoclonal antibody. Using this antibody MMP-9 is captured from biological fluids or tissue culture media, and MMP-activity of both active and latent MMP-9 can be analysed. We determined the gelatinase-B (MMP-9) activity present in saliva from patients with Sjögren's syndrome. Using a general gelatinase assay with radioactively-labeled gelatinated collagen it was observed that gelatinase activity was slightly, though not significantly, increased in patients: general gelatinase activity in patients versus healthy controls: 17.0 +/- 4.9 vs 12.2 +/- 2.5 x 10(4) cpm/ml (p > 0.05, and 44.0 (4.0 vs 36.1 +/- 1.9 x 10(4) cpm/ml (p > 0.05), for active and latent gelatinase, respectively. However, using the immunocapture activity assay (using modified urokinase) specifically MMP-9 activity was measured, which was significantly increased in saliva from patients compared to 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 = 0.01). This assay, measuring MMP-9 activity using modified pro-urokinase as a substrate can easily be adapted for the specific detection of the various members of the MMP-family or other difficult to measure proteases, in a format that can be used for high throughput screening of compounds or samples.

Collagenase-3 (MMP-13) and its activators in rheumatoid arthritis: localization in the pannus-hard tissue junction and inhibition by alendronate.

The hypothesis of the present work was that the pannus tissue overlying the articular hard tissues has an aggressive phenotype and contains the newly discovered collagenase-3 and its endogenous inducers and activators. We therefore analyzed the eventual presence of collagenase-3 and its regulation at the pannus-cartilage junction. Collagenase-3 mRNA (in situ hybridization) and enzyme protein (ABC and immunofluorescence staining) were found in the pannocytes in the pannus-hard tissue junction. Inflammatory round cells associated with the critical interface contained TNF-alpha and IL-1beta. These cytokines induced collagenase-3 secretion in cultured rheumatoid synovial fibroblasts. Procollagenase-3 activators, stromelysin-1, 72 kDa type IV collagenase/gelatinase and membrane-type 1-MMP, were also found in the pannus-hard tissue junction. Active collagenase-3 was inhibited with alendronate (IC50 = 500-750 microM). Collagenase-3, due to its substrate profile and local synthesis in a milieu favoring its activation, might play a major role in the degradation of cartilage type II and bone type I collagens. Alendronate, at concentrations attainable in vivo, is able to inhibit collagenase-3. This might offer an option to control collagenase-3-mediated tissue destruction in rheumatoid arthritis.

Matrix metalloproteinase (MMP)-9 type IV collagenase/gelatinase implicated in the pathogenesis of Sjögren's syndrome.

Type IV collagenases/gelatinases (matrix metalloproteinases MMP-2 and MMP-9) in labial salivary glands (LSG) and saliva in Sjögren's syndrome (SS) and healthy controls were studied. Zymograms and Western blots disclosed that SS saliva contained 92/82 kD MMP-9/type IV collagenase duplex. Specific activity measurement disclosed 53.1+/-9.8 U/mg protein MMP-9 in SS compared to 16.5+/-2.6 U/mg in healthy controls (p=0.01). MMP-2 did not differ between SS and controls. In SS salivary glands, MMP-2 and MMP-9 were also expressed, in addition to stromal fibroblasts and occasional infiltrating neutrophils, respectively, in acinar end piece cells. In addition, an effective proMMP-9 activator, human trypsin-2 (also known as tumor-associated trypsin-2 or TAT-2), was found in acinar end piece cells and in saliva. Interestingly, proteolytically processed MMP-9 was found in saliva (vide supra), and in vivo activated MMP-9 was significantly higher in SS than in controls (p=0.002). LSGs, particularly in SS, were characterized ultrastructurally by areas containing small cytoplasmic vesicles in the basal parts of the epithelial cells associated with areas of disordered and thickened basal lamina. Based on our results, we conclude here that SS saliva contains increased concentrations of MMP-9, which is of glandular origin in part. Pro MMP-9 is to a large extent proteolytically activated. This is probably mediated by the most potent pro MMP-9 activator found in vivo thus far, namely trypsin-2. Therefore, the MMP 9/trypsin-2 cascade may be responsible for the increased remodelling and/or structural destruction of the basement membrane scaffolding in salivary glands in SS. Due to the role of basal lamina as an important molecular sieve and extracellular matrix-cell signal, these pathological changes may contribute to the pathogenesis of the syndrome.

Time-resolved fluorescence studies on the dihydrolipoyl transacetylase (E2) component of the pyruvate dehydrogenase complex from Azotobacter vinelandii.

The dihydrolipoyl transacetylase (E2) component of A. vinelandii PDC and its lipoyl domain shows similar dynamic properties as revealed with fluorescence anisotropy decay of lipoyl-bound IAANS. The lipoyl domain (32.6 kDa), containing three almost identical subdomains shows a mode of rotation characteristic for a protein of about 30 kDa. A similar rotation is found in E2, indicating an independent rotational mobility of the whole domain in the multimeric E2 core (1.6 MDa). No independent rotation of a single lipoyl subdomain (10 kDa) is observed. The E1 component, in contrast to the E3 component, shows interaction with the lipoyl domain.

Mobile sequences in the pyruvate dehydrogenase complex, the E2 component, the catalytic domain and the 2-oxoglutarate dehydrogenase complex of Azotobacter vinelandii, as detected by 600 MHz 1H-NMR spectroscopy.

600 MHz 1H-NMR spectroscopy demonstrates that the pyruvate dehydrogenase complex of Azotobacter vinelandii contains regions of the polypeptide chain with intramolecular mobility. This mobility is located in the E2 component and can probably be ascribed to alanine-proline-rich regions that link the lipoyl subdomains to each other as well as to the E1 and E3 binding domain. In the catalytic domain of E2, which is thought to form a compact, rigid core, also conformational flexibility is observed. It is conceivable that the N-terminal region of the catalytic domain, which contains many alanine residues, is responsible for the observed mobility. In the low-field region of the 1H-NMR spectrum of E2 specific resonances are found, which can be ascribed to mobile phenylalanine, histidine and/or tyrosine residues which are located in the E1 and E3 binding domain that links the lipoyl domain to the catalytic domain. In the 1H-NMR spectrum of the intact complex, these resonances cannot be observed, indicating a decreased mobility of the E1 and E3 binding domain.

Role of fibrin and plasminogen activators in repair-associated angiogenesis: in vitro studies with human endothelial cells.

Angiogenesis, the formation of new blood vessels from existing ones, plays a central role in development and in a number of pathological conditions. Tissue repair-associated angiogenesis usually involves cell invasion into a fibrin structure and the presence of inflammatory cells. In this chapter the role of plasminogen activators in the dissolution of fibrin and the invasion of endothelial cells into a fibrin matrix is described. Tissue-type plasminogen activator is stored in endothelial cells and can be released acutely into the vessel lumen upon stimulation of the endothelium to activate fibrinolysis and to prevent fibrin deposition. At the basolateral side of the cell, urokinase-type plasminogen activator (uPA) bound to a specific cellular receptor is involved in the proteolytic modulation of matrix proteins and cell-matrix interaction. The cytokine tumor necrosis factor-alpha (TNF-alpha) cooperates with the angiogenic factors basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in inducing human microvascular endothelial cells in vitro to invade a three dimensional fibrin matrix and to form capillary-like tubular structures. The formation of these capillary-like tubules requires cell-bound uPA activity.

Matrix-metalloproteinase activity in first trimester placental bed biopsies in further complicated and uncomplicated pregnancies.

UNLABELLED: Trophoblast invasion is partly regulated by matrix-metalloproteinases (MMPs). Aberrations in MMP-activity in early pregnancy are thought to play a role in the pathophysiology of pregnancy associated conditions like pre-eclampsia and intra-uterine growth restriction (IUGR). A direct relation however, has not been published. We tested the hypothesis that MMP activity in the decidua is compromised in the first trimester of pregnancies, which are complicated by hypertensive disorders or IUGR in later pregnancy. During chorionic villus biopsy, decidua is microscopically separated from the villi and stored. A selection of pregnancies complicated by pre-eclampsia or HELLP-syndrome or IUGR was made, with two matched controls each. Zymography was performed to identify the presence of MMPs, and subsequently immunohistochemistry for MMP-2 and -9 and cytokeratin 7 to examine differences between cases and controls. Next, a specific immuno-capture assay was used to determine the activity of MMP-1, -2, -3, -8, -9, and 13, total as well as active. Although presence of MMP-2 and MMP-9 was found, which corresponded with the immunohistochemistry, no significant differences could be demonstrated between activity of total MMP-2 and total MMP-9 in complicated and uncomplicated pregnancies. Activity of MMP-1, -3, -8 and -13 could not be detected. IN CONCLUSION: our study confirms the presence of MMP-2 and -9 in first trimester placental bed biopsies, but does not support the current concept of deranged MMP-activity in early pregnancy in further complicated pregnancies.

A patient with a VEGF and endostatin producing gastrointestinal autonomic nerve tumour.

Tumour associated neovascularisation has been characterised as chaotic and insufficient. This report details the results of the analysis of angiogenic factors in tumour cyst fluid, pleural fluid, and blood from a patient with a gastrointestinal autonomic nerve tumour. The tumour produced vascular endothelial growth factor and endostatin in large quantities, which may explain the dysfunctional angiogenesis and tendency to bleeding seen in this tumour type.

MMP-9 activity in urine from patients with various tumors, as measured by a novel MMP activity assay using modified urokinase as a substrate.

Matrix metalloproteinases (MMPs) play an important role in many pathologic processes, but their activities are difficult to determine since no simple specific and/or chromogenic substrate exists. We have developed a novel MMP activity assay using a modified urokinase as a substrate. Protein engineering enabled the plasmin activation site in this urokinase to be substituted by a specific activation site recognized by MMPs. In this way the MMP activity can be monitored via urokinase activity as measured by a simple chromogenic assay. The assay was made specific for MMP-9 by a capture step using MMP-9-specific antibodies that do not interfere with MMP-activity. This assay monitors the amount of active enzyme as well as the latent, but potentially active proform. Using this assay the levels of MMP-9 were investigated in urine from patients with various kinds of carcinoma. High levels of both active and latent MMP-9 were detected in urine from patients with carcinoma of the bladder, whereas hardly any activity was observed in urine from healthy controls. MMP-9 in urine was present in its intact form. Surprisingly, MMP-9 was also increased in the urine of patients with nonurogenital carcinoma. Therefore, measurement of urinary MMP-9 activity levels may be a convenient diagnostic tool for various types of carcinoma.

MMP inhibition and downregulation by bisphosphonates.

Bisphosphonates are a group of drugs capable of inhibiting bone resorption, and are thus used for the treatment of bone diseases, such as Paget's disease, osteoporosis, and for bone metastases of malignant tumors. Their primary cellular target is considered to be the osteoclast. The molecular mechanisms responsible for the downregulation of bone resorption by bisphosphonates have remain unclear. We have discovered that various matrix metalloproteinases (MMPs) are inhibited in vitro by several bisphosphonates. This novel finding may, in part, explain the efficacy of bisphosphonates in their current indications in humans. In enzyme activity tests using purified and recombinant enzymes, we have observed the inhibition of MMP-1, -2, -3, -7, -8, -9, -12, -13, and -14 by clondronate, alendronate, pamidronate, zolendronate, nedrinate, and clodrinate. The IC50s range from 50 to 150 microM. We have also shown that clodronate can downregulate the expression of MT1-MMP protein and mRNA in several cell lines. Additionally, several bisphosphonates decrease the degree of invasion of malignant melanoma (C8161) and fibrosarcoma (HT1080) cells through artificial basement membrane (Matrigel) in cell cultures at IC50s of 50-150 microM and below. Having low toxicity and proven to be well tolerated after several years in human use, bisphosphonates have the potential to become one of the main MMP-inhibitors for MMP-related human soft and hard tissue-destructive diseases in the near future.

Regulation of MMP-9 (gelatinase B) in activated human monocyte/macrophages by two different types of bisphosphonates.

Metalloproteinases (MMP), particularly MMP-9 produced by the intratumor monocyte/macrophages, play an important role in tumor invasion and metastases. Recent clinical trials in patients with primary breast cancer suggest that bisphosphonates (BP), above all clodronate, may reduce bone metastases. The aim of the present study was to evaluate whether the effects of BPs on cancer dissemination include inhibition of MMP-9 production in human monocyte/macrophages. The effects of clodronate and pamidronate on the MMP-9 expression in and secretion from stimulated human monocyte/macrophages were measured using quantitative reverse transcriptase - polymerase chain reaction (RT-PCR) and enzyme-linked immunoadsorbent assay (ELISA), respectively. The MMP-9 mRNA levels remained relatively stable in the presence of clodronate. In contrast, pamidronate at 30 microM-300 microM increased the mRNA levels 5- to 10-fold. MMP-9 secretion was dose-dependently down-regulated by clodronate whereas pamidronate at 30 microM induced a 50% increase on MMP-9 secretion (p < 0.05), followed by a down-regulation at higher concentrations. The results suggest that MMP-9 is differentially regulated at mRNA and enzyme protein level by BPs, which affect ATP-dependent intracellular enzymes (clodronate) or post-translational modification of GTPases (pamidronate). These findings may have implications for the therapeutic use of these compounds.

Active MMPs captured by alpha 2 macroglobulin as a marker of disease activity in rheumatoid arthritis.

OBJECTIVE: The aim of the present study was to analyze alpha 2 Macroglobulin/MMP (alpha 2M/MMP) complex formation and to investigate whether MMP activity in alpha 2M/MMP complexes in serum can be used as a disease marker in rheumatoid arthritis (RA). METHODS: High and low molecular weight (H/LMW) substrates and inhibitors and size exclusion were used to analyze alpha 2M/MMP complex formation. LMW fluorogenic substrates were used to quantify the level of MMPs in alpha 2M/MMP complexes in the serum of RA patients and healthy controls. RESULTS: Active MMPs were fully inhibited by LMW inhibitor BB94 in the presence of alpha 2M, whereas no inhibition was achieved by HMW inhibitor TIMP-1. Size exclusion analysis showed alpha 2M/MMP complex formation in buffer and in normal plasma spiked with activated MMPs, which indicated alpha 2M/MMP complex formation in the systemic circulation. MMP activity in alpha 2M/MMP complexes in the serum of RA patients was significantly higher than in the serum of healthy controls (P < 0.001). MMP activity levels in the serum of RA patients were correlated with ESR (r = 0.72, P < 0.001). CONCLUSION: In the systemic circulation of RA patients, active MMPs form complexes with alpha 2M and can be detected using LMW fluorogenic substrates. MMP activity measurements in serum allow discrimination between RA patients and healthy controls and provide a new tool for the assessment of the disease process in RA.

Imbalance of RANKL/RANK/OPG system in interface tissue in loosening of total hip replacement.

In the differentiation of osteoclasts the differentiation factor (RANKL) interacts with the receptor activator of NF-kappaB (RANK) in a direct cell-to-cell contact between osteoblast and (pre)osteoclast. This is inhibited by soluble osteoprotegerin (OPG). The mRNA levels of both RANKL (p < 0.01) and RANK (p < 0.05) were high in peri-implant tissue and RANKL+ and RANK+ cells were found in such tissue. Double labelling also disclosed soluble RANKL bound to RANK+ cells. We were unable to stimulate fibroblasts to express RANKL in vitro, but monocyte activation with LPS gave a fivefold increase in RANK mRNA levels. In contrast to RANKL and RANK expression in peri-implant tissue, expression of OPG was restricted to vascular endothelium. Endothelial cell OPG mRNA levels were regulated by TNF-alpha and VEGF, but not by hypoxia. It is concluded that activated cells in the interface tissue overproduce both RANKL and RANK and they can interact without interference by OPG.