Modulation of adipose tissue expression of murine matrix metalloproteinases and their tissue inhibitors with obesity

The potential role of the matrix metalloproteinase (MMP) system in the pathophysiology of the adipose tissue was investigated in a mouse model of nutritionally induced obesity. mRNA levels of 16 MMPs and 4 tissue inhibitors of MMPs (TIMPs) were measured by semiquantitative RT-PCR in adipose tissue isolated from mice maintained for 15 weeks on a standard or high-fat diet. In mice on standard diet, with the exception of MMP-8, all MMP and TIMP transcripts were detected in both gonadal and subcutaneous depots. In obese mice, the expression of MMP-3, -11, -12, -13, and -14 and TIMP-1 mRNAs was upregulated, whereas that of MMP-7, -9, -16, and -24 and TIMP-4 was downregulated. Most MMP and TIMP mRNAs were expressed at higher levels in stromal-vascular cells than in mature adipocytes. Analysis of adipose tissue by in situ fluorescent zymography revealed MMP-dependent proteolytic activities, demonstrating the presence of active MMPs in the intact tissue. In vitro conversion of adipogenic 3T3-F442A cells into mature adipocytes was associated with substantial modulations of MMP and TIMP expression. Moreover, this in vitro adipogenesis was reduced in the presence of a synthetic MMP inhibitor. Thus, the adipose tissue expresses a large array of MMPs and TIMPs, which modulate adipocyte differentiation

Regulation of procollagen amino-propeptide processing during mouse embryogenesis by specialization of homologous ADAMTS proteases: insights on collagen biosynthesis and dermatosparaxis

Mutations in ADAMTS2, a procollagen amino-propeptidase, cause severe skin fragility, designated as dermatosparaxis in animals, and a subtype of the Ehlers-Danlos syndrome (dermatosparactic type or VIIC) in humans. Not all collagen-rich tissues are affected to the same degree, which suggests compensation by the ADAMTS2 homologs ADAMTS3 and ADAMTS14. In situ hybridization of Adamts2, Adamts3 and Adamts14, and of the genes encoding the major. brillar collagens, Col1a1, Col2a1 and Col3a1, during mouse embryogenesis, demonstrated distinct tissue-specific, overlapping expression patterns of the protease and substrate genes. Adamts3, but not Adamts2 or Adamts14, was co-expressed with Col2a1 in cartilage throughout development, and with Col1a1 in bone and musculotendinous tissues. ADAMTS3 induced procollagen I processing in dermatosparactic. broblasts, suggesting a role in procollagen I processing during musculoskeletal development. Adamts2, but not Adamts3 or Adamts14, was co-expressed with Col3a1 in many tissues including the lungs and aorta, and Adamts2(-/-) mice showed widespread defects in procollagen III processing. Adamts2(-/-) mice had abnormal lungs, characterized by a decreased parenchymal density. However, the aorta and collagen fibrils in the aortic wall appeared normal. Although Adamts14 lacked developmental tissue-specific expression, it was co-expressed with Adamts2 in mature dermis, which possibly explains the presence of some processed skin procollagen in dermatosparaxis. The data show how evolutionarily related proteases with similar substrate preferences may have distinct biological roles owing to tissue specific gene expression, and provide insights into collagen biosynthesis and the pathobiology of dermatosparaxis

MMP-9 regulates both positively and negatively collagen gel contraction - A nonproteolytic function of MMP-9

peer reviewedaudience: researcher, professionalObjective: Constrictive remodeling accounts for lumen loss in postangioplasty restenosis. Matrix metalloproteinase-9 (MMP-9) has been shown to prevent constrictive remodeling in vivo. To investigate potential mechanisms for this observation, we investigated the role of MMP-9 in smooth muscle cell (SMC)-mediated collagen gel contraction, an in vitro model of constrictive remodeling. Methods: Fischer rat SMCs were stably transfected with a construct-expressing rat-MMP-9 under the control of a tetracycline (Tet)-off promoter. SMCs were seeded in type 1 collagen gels (2.4 mg/ml) in the presence or not of tetracycline (1 mu g/ml), and gel contraction was defined as the percentage of retraction of the collagen gel. The depletion of MMP-9 was obtained by using siRNA targeting MMP-9 mRNA or a blocking antibody. Results: Gel contraction was significantly reduced at all times when MMP-9 was overexpressed (Tet-) as compared with the control condition (Tet+). However, MMP-9 depletion of control (Tet+) SMCS (using siRNA or antibody) also inhibited gel contraction. To resolve the apparent discrepancy and determine if MMP-9 exerts a dose-dependent biphasic effect on gel contraction, conditioned medium and purified rat-MMP-9 were prepared. Gel contraction was significantly increased by addition of 0.8 mg/ml of MMP-9, while high concentrations of MMP-9 (>= 100 mg/ml) inhibited contraction. The addition of BB94 and TIMP-1 did not alter the inhibitory or stimulatory effect of MMP-9. Conclusions: Our data Suggest that MMP-9, independent of its proteolytic function, has a biphasic effect on SMC-mediated collagen gel contraction. Understanding the different roles of MMP-9 Should allow the development of better therapeutic strategies for restenotic vascular disease. (c) 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved

Down-Regulation of Vascular Endothelial Growth Factor by Tissue Inhibitor of Metalloproteinase-2: Effect on in Vivo Mammary Tumor Growth and Angiogenesis

The tissue inhibitor of metalloproteinases-2 (TIMP-2) has at least two independent functions, i.e., regulation of matrix metalloproteinases and growth promoting activity. We investigated the effects of TIMP-2 overexpression, induced by retroviral mediated gene transfer, on the in vivo development of mammary tumors in syngeneic mice inoculated with EF43.fgf-4 cells. The EF43.fgf-4 cells established by stably infecting the normal mouse mammary EF43 cells with a retroviral expression vector for the fgf-4 oncogene, are highly tumorigenic and overproduce vascular endothelial growth factor (VEGF). Despite a promotion of the in vitro growth rate of EF43.fgf-4 cells overexpressing timp-2, the in vivo tumor growth was delayed. At day 17 post-cell injection, the volume of tumor derived from TIMP-2-overexpressing cells was reduced by 80% as compared with that obtained with control cells. Overexpression of TIMP-2 was associated with a down-regulation of VEGF expression in vitro and in vivo, a reduction of vessel size, density, and blood supply in the induced tumors. In addition, TIMP-2 completely inhibited the angiogenic activity of EF43.fgf-4 cell-conditioned medium in vitro using a rat aortic ring model. Our findings suggest that overexpression of TIMP-2 delays growth and angiogenesis of mammary carcinoma in vivo and that down-regulation of VEGF expression may play an important role in this TIMP-2-mediated antitumoral and antiangiogenic effects. Finally the in vivo delivery of TIMP-2, as assessed by i.v. injection of recombinant adenoviruses vectors, significantly reduced the growth of the EF43.fgf-4-induced tumors. This effect of TIMP-2 was shown to be equally comparable with that of angiostatin, a known potent inhibitor of angiogenesis

Gene expression and matrix turnover in overused and damaged tendons

Chronic, painful conditions affecting tendons, frequently known as tendinopathy, are very common types of sporting injury. The tendon extracellular matrix is substantially altered in tendinopathy, and these changes are thought to precede and underlie the clinical condition. The tendon cell response to repeated minor injuries or “overuse” is thought to be a major factor in the development of tendinopathy. Changes in matrix turnover may also be effected by the cellular response to physical load, altering the balance of matrix turnover and changing the structure and composition of the tendon. Matrix turnover is relatively high in tendons exposed to high mechanical demands, such as the supraspinatus and Achilles, and this is thought to represent either a repair or tissue maintenance function. Metalloproteinases are a large family of enzymes capable of degrading all of the tendon matrix components, and these are thought to play a major role in the degradation of matrix during development, adaptation and repair. It is proposed that some metalloproteinase enzymes are required for the health of the tendon, and others may be damaging, leading to degeneration of the tissue. Further research is required to investigate how these enzyme activities are regulated in tendon and altered in tendinopathy. A profile of all the metalloproteinases expressed and active in healthy and degenerate tendon is required and may lead to the development of new drug therapies for these common and debilitating sports injuries

The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) family

The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) enzymes are secreted, multi-domain matrix-associated zinc metalloendopeptidases that have diverse roles in tissue morphogenesis and patho-physiological remodeling, in inflammation and in vascular biology. The human family includes 19 members that can be sub-grouped on the basis of their known substrates, namely the aggrecanases or proteoglycanases (ADAMTS1, 4, 5, 8, 9, 15 and 20), the procollagen N-propeptidases (ADAMTS2, 3 and 14), the cartilage oligomeric matrix protein-cleaving enzymes (ADAMTS7 and 12), the von-Willebrand Factor proteinase (ADAMTS13) and a group of orphan enzymes (ADAMTS6, 10, 16, 17, 18 and 19). Control of the structure and function of the extracellular matrix (ECM) is a central theme of the biology of the ADAMTS, as exemplified by the actions of the procollagen-N-propeptidases in collagen fibril assembly and of the aggrecanases in the cleavage or modification of ECM proteoglycans. Defects in certain family members give rise to inherited genetic disorders, while the aberrant expression or function of others is associated with arthritis, cancer and cardiovascular disease. In particular, ADAMTS4 and 5 have emerged as therapeutic targets in arthritis. Multiple ADAMTSs from different sub-groupings exert either positive or negative effects on tumorigenesis and metastasis, with both metalloproteinase-dependent and -independent actions known to occur. The basic ADAMTS structure comprises a metalloproteinase catalytic domain and a carboxy-terminal ancillary domain, the latter determining substrate specificity and the localization of the protease and its interaction partners; ancillary domains probably also have independent biological functions. Focusing primarily on the aggrecanases and proteoglycanases, this review provides a perspective on the evolution of the ADAMTS family, their links with developmental and disease mechanisms, and key questions for the future

Distinct pathways in the over-expression of matrix metalloproteinases in human fibroblasts by relaxation of mechanical tension.

peer reviewedaudience: researcherThe aim of the work was to analyze, on a comparative basis, the signaling pathways operating in the regulation of a panel of matrix metalloproteinases (MMP) expressed by human dermal fibroblasts submitted to mechanical stress relaxation by cytochalasin D (CD) and in a retracting collagen gel (RCG). The mRNA steady-state level of MMPs was measured by a quantitative RT-PCR procedure using a synthetic RNA as internal standard. In monolayer, most MMPs were barely detected, except MMP-2. Disruption of the actin stress fibers by CD induced a moderate increase of MMP-2 mRNA and a much larger stimulation of MMP-3, -9, -13 and -14 mRNAs. In RCG, a significant up-regulation of these MMPs was also observed although to a lower extent than in CD-treated monolayers. Among the investigated MMPs, the MMP-8 and -11 were not reproducibly detected. MMP-2 was processed to its active form both by CD and in RCG. The CD-induced up-regulation of gene expression was largely repressed by blocking protein synthesis by cycloheximide for all the MMPs, by inhibiting the tyrosine-kinases of the src family by herbimycin A for all MMPs, except MMP-2, and by inhibiting the TPA-inducible PKC isoforms by bisindoyl maleimide for all MMPs, except MMP-14. The up-regulation induced by stress relaxation in RCG was protein synthesis-dependent for MMP-2 and MMP-13, tyrosine kinases-dependent for MMP-3 and MMP-13, as previously described for MMP-1. Inhibiting TPA-inducible PKC did not affect any MMP in RCG except MMP-13, which was strongly induced. The processing of MMP-2 was tyrosine kinases-dependent but PKC-independent. Inhibitors of the ERK1,2 and p38 MAP kinases pathways diversely affected the MMPs expression. Inhibiting the Rho-kinase activity by Y-27632 was inactive. These results point to the potent regulation operated by the status of the cytoskeleton on the cell phenotype, and to distinct regulatory pathways involved in the control of different MMPs expression

Changes in matrix gene and protein expressions after single or repeated exposure to one minimal erythemal dose of solar-simulated radiation in human skin in vivo

peer reviewedaudience: researcher, professionalDamage to the skin extracellular matrix (ECM) is the hallmark of long-term exposure to solar UV radiation. The aim of our study was to investigate the changes induced in unexposed human skin in vivo after single or repeated (five times a week for 6 weeks) exposure to I minimal erythemal dose (MED) of UV solar-simulated radiation. Morphological and biochemical analyses were used to evaluate the structural ECM components and the balance between the degrading enzymes and their physiologic inhibitors. A three-fold increase in matrix metalloproteinase 2 messenger RNA (mRNA) (P < 0.02, unexposed versus exposed) was observed after both single and repeated exposures. Fibrillin 1 mRNA level was increased by chronic exposure (P < 0.02) and unaltered by a single MED. On the contrary, a single MED significantly enhanced mRNA levels of interleukin-la (IL-1alpha), IL-1beta (P < 0.02) and plasminogen activator inhibitor-1 (P < 0.05). Immunohistochemistry demonstrated a significant decrease in Type-I procollagen localized just below the dermal-epidermal junction in both types of exposed sites. At the same location, the immunodetected tenascin was significantly enhanced, whereas a slight increase in Type-III procollagen deposits was also observed in chronically exposed areas. Although we were unable to observe any change in elastic fibers in chronically exposed buttock skin, a significant increase in lysozyme and alpha-1 antitrypsin deposits on these fibers was observed. These results demonstrate the existence of a differential regulation, after chronic exposure compared with an acute one, of some ECM components and inflammatory mediators