Matrix metalloproteinases MMP-2 and MMP-9 of wound and burn exudates and their effect on extracellular matrix proteins

The dynamics of matrix metalloproteinases (MMP), as well as of fibronectin concentration in wound and burn fluids was traced. The wound fluid proteolytic activity was studied by gelatin zymography method. The data on degradation of fibronectin and various laminin isoforms by wound fluid proteases show that laminin-1, laminin-2/4 and fibronectin were degraded by wound fluid into small fragments. Remodelling of extracellular matrix proteins occurs. Dynamics of MMP-2 and MMP-9 content in wound or burn fluids as well as that of adhesive protein fibronectin content could be used as a base for development of method of controlling the extracellular matrix remodelling process.     

Temporal study of the activity of matrix metalloproteinases and their endogenous inhibitors during wound healing

The restoration of functional connective tissue is a major goal of the wound healing process. This regenerative event requires the deposition and accumulation of collagenous and noncollagenous matrix molecules as well as the remodelling of extracellular matrix (ECM) by matrix metalloproteinases (MMPs). In this study, we have utilized substrate gel electrophoresis, radiometric enzyme assays, and Western blot analyses to determine the temporal pattern of appearance and activity of active and latent MMPs and their inhibitors during the entire healing process in a partial thickness wound model. Through the use of substrate gel electrophoresis, we studied the appearance of proteolytic bands whose molecular weight was consistent with their being members of the MMP family of enzymes. Proteolytic bands whose molecular weight is consistent with both the active and latent forms of MMP-2 (72 kDa, Type IV gelatinase) were detected in wound fluid of days 1–7 after wounding. The number of active MMP-2 species detectable in wound fluid was greatest during days 4–6 after wounding. The most prominent proteolytic band detected each day migrated with a molecular weight consistent with it being the latent form of MMP-9 (92 kDa, Type V pro-collagenase). In contrast to MMP-2, the active form of this enzyme was never detected. The presence of MMP-1 (interstitial collagenase) was detected by immunoblot in the wound fluid from days 1–6 post-injury. Using a radiometric enzyme assay for collagenase inhibitory activity we have also determined the time course of activity of endogenous matrix metalloproteinase inhibitors. We have correlated these data to the known cellular events occurring in the wound during this time period as well. This study establishes a prototypical pattern of MMP appearance in normal wound healing. It may also provide potential intervention sites for the therapeutic use of inhibitors of aberrant MMP activities which characterize chronic wounds. © 1996 Wiley-Liss, Inc.     

Development of immunosensors for direct detection of three wound infection biomarkers at point of care using electrochemical impedance spectroscopy

A method for label-free, electrochemical impedance immunosensing for the detection and quantification of three infection biomarkers in both buffer and directly in the defined model matrix of mock wound fluid is demonstrated. Triggering Receptor-1 Expressed on Myeloid cells (TREM-1) and Matrix MetalloPeptidase 9 (MMP-9) are detected via direct assay and N-3-oxo-dodecanoyl-l-HomoSerineLactone (HSL), relevant in bacterial quorum sensing, is detected using a competition assay. Detection is performed with gold screen-printed electrodes modified with a specific thiolated antibody. Detection is achieved in less than 1h straight from mock wound fluid without any extensive sample preparation steps. The limits of detection of 3.3 pM for TREM-1, 1.1 nM for MMP-9 and 1.4 nM for HSL are either near or below the threshold required to indicate infection. A relatively large dynamic range for sensor response is also found, consistent with interaction between neighbouring antibody-antigen complexes in the close-packed surface layer. Together, these three novel electrochemical immunosensors demonstrate viable multi-parameter sensing with the required sensitivity for rapid wound infection detection directly from a clinically relevant specimen.     

Transforming growth factor-beta1 increases airway wound repair via MMP-2 upregulation: a new pathway for epithelial wound repair?

In vivo, transforming growth factor (TGF)-beta1 and matrix metalloproteinases (MMPs) present at the site of airway injury are thought to contribute to epithelial wound repair. As TGF-beta1 can modulate MMP expression and MMPs play an important role in wound repair, we hypothesized that TGF-beta1 may enhance airway epithelial repair via MMPs secreted by epithelial cells. We evaluated the in vitro influence of TGF-beta1 on wound repair in human airway epithelial cells cultured under conditions allowing differentiation. The results showed that TGF-beta1 accelerated in vitro airway wound repair, whereas MMP inhibitors prevented this acceleration. In parallel, we examined the effect of TGF-beta1 on the expression of MMP-2 and MMP-9. TGF-beta1 induced a dramatic increase of MMP-2 expression with an increased steady-state level of MMP-2 mRNA, contrasting with a slight increase in MMP-9 expression. To confirm the role of MMP-2, we subsequently evaluated the effect of MMP-2 on in vitro airway wound repair and demonstrated that the addition of MMP-2 reproduced the acceleration of wound repair induced by TGF-beta1. These results strongly suggest that TGF-beta1 increases in vitro airway wound repair via MMP-2 upregulation. It also raises the issue of a different in vivo biological role of MMP-2 and MMP-9 depending on the cytokine microenvironment.     

Possibility of predicting rat wound epithelization by changes in matrix metalloproteinases activities in wound exudate

The engraftment of a free skin graft introduced into an unhealing wound as a source of epithelization in combination with a transplantation of a dermal equivalent was studied in rats. The course of wound healing was estimated by changes in the activity levels of metalloproteinases (MMPs) in wound exudates. It was shown that the results of skin-graft transplantation could be predicted by monitoring changes in wound exudates MMP-2 and MMP-9 activities. It was found that engraftment of the skin graft occurred at intermediate activity values of MMP-2 and MMP-9 in the wound exudates, whereas their low and high activities correspond to lysis of the transplanted skin graft.     

Mice that lack matrix metalloproteinase-9 display delayed wound healing associated with delayed reepithelization and disordered collagen fibrillogenesis

Matrix metalloproteinase- (MMP-9) is involved in processes that occur during cutaneous wound healing such as inflammation, matrix remodeling, and epithelialization, To investigate its role in healing, full thickness skin wounds were made in the dorsal region of MMP-9-null and control mice and harvested up to 14 days post wounding. Gross examination and histological and immunohistochemical analysis indicated delayed healing in MMP-9-null mice. Specifically, MMP-9-null wounds displayed compromised reepithelialization and reduced clearance of fibrin clots. In addition, they exhibited abnormal matrix deposition, as evidenced by the irregular alignment of immature collagen fibers. Despite the presence of matrix abnormalities, MMP-9-null wounds displayed normal tensile strength. Ultrastructural analysis of wounds revealed the presence of large collagen fibrils, some with irregular shape. Keratinocyte proliferation, inflammation, and angiogenesis were found to be normal in MMP-9-null wounds. In addition, VEGF levels were similar in control and MMP-9-null wound extracts. To investigate the importance of MMP-9 in wound reepithelialization we tested human and murine keratinocytes in a wound migration assay and found that antibody-based blockade of MMP-9 function or MMP-9 deficiency retarded migration. Collectively, our observations reveal defective healing in MMP-9-null mice and suggest that MMP-9 is required for normal progression of wound closure.     

Loss of epidermal MMP-14 expression interferes with angiogenesis but not with re-epithelialization

Synthesis and activation of matrix metalloproteinases during wound healing are important for remodeling the extracellular matrix and modulating various cellular functions. The membrane-type 1 matrix metalloproteinase (MMP-14) has been shown to play a key role during these processes. To analyze the function of epidermal-derived MMP-14 during skin repair we generated mice lacking MMP-14 expression in the epidermis (MMP-14(ep-/-)). These mice displayed overall normal skin morphology and epidermal differentiation patterns. Wound repair in MMP-14(ep-/-) followed the same kinetics as in wild type mice (MMP-14(ep+/+)), and infiltration of neutrophils, leukocytes, and macrophages into the wound site was comparable. Microscopic analysis showed no altered re-epithelialization in the absence of epidermal MMP-14. Furthermore, epidermal differentiation at the end of the repair process and scar formation was normal. However, at day 14 post wounding, sustained angiogenesis was observed in MMP-14(ep-/-) mice in contrast to control mice. Interestingly, decreased levels of endostatin were detected in wound lysates of MMP-14(ep-/-) mice as well as in cultured keratinocytes. Taken together, these data indicate that MMP-14 expression in keratinocytes is dispensable for skin homeostasis and repair, but plays a crucial role in the epidermal-dermal crosstalk leading to modulation of vessel density.     

Overexpression of TIMP-1 under the MMP-9 promoter interferes with wound healing in transgenic mice

We have generated transgenic mice harboring the murine matrix metalloproteinase 9 (MMP-9) promoter cloned in front of human TIMP-1 cDNA. The transgenic mice were viable and fertile and exhibited normal growth and general development. During wound healing the mice were shown to express human TIMP-1 in keratinocytes that normally express MMP-9. However, the healing of skin wounds was significantly retarded with slow migration of keratinocytes over the wound in transgenic mice. In situ zymography carried out on wound tissues revealed total blockage of gelatinolytic activity (i.e., MMP-9 and MMP-2). The results confirm studies with MMP-9 knockout mice showing that MMP-9 is not essential for general development, but they also demonstrate an important role of keratinocyte MMP-9, as well that of other keratinocyte MMPs that are inhibited by TIMP-1, in wound healing. The transgenic mice generated in this study provide a model for the role of MMPs in MMP-9-producing cells in other challenging situations such as bone fracture recovery and cancer invasion.The expert technical assistance of M. Jarva, L. Ollitervo, S. Kangas, and R. Jokisalo is gratefully acknowledged. This work was supported in part by grants from the Finnish Academy of Science, the Swedish Cancer Foundation, the Novo Nordisk Foundation and EC contract QLG1-CT-2000-01131 (K.T.), the Finnish Dental Society Apollonia and the Northern Finland Cancer Foundation (M.P.), as well as the K. Albin Johansson Foundation and the Einar and Karin Stroems Foundation (E.P.)     

Thymosin beta4 promotes matrix metalloproteinase expression during wound repair

Immobilized patients, diabetics, and the elderly suffer from impaired wound healing. The 43-amino acid angiogenic peptide thymosin beta4 (Tbeta4) has previously been found to accelerate dermal wound repair in rats, aged mice, and db/db diabetic mice. It also promotes corneal repair in both normal rats and mice. Because proteinases are important in wound repair, we hypothesized that Tbeta4 may regulate matrix metalloproteinase (MMP) expression in cells that are involved in wound repair. Analysis by RT-PCR of whole excised mouse dermal wounds on days 1, 2, and 3 after wounding showed that Tbeta4 increased several metalloproteinases, including MMP-2 and -9 expression by several-fold over control on day 2 after wounding. We further analyzed the metalloproteinases secreted in response to exogenous Tbeta4 by cells normally present in the wound. Western blot analysis of cultured keratinocytes, endothelial cells, and fibroblasts that were treated with increasing concentrations of Tbeta4 showed increases in the levels of MMP-1, -2, and -9 in a cell-specific manner. Tbeta4 also enhanced the secretion of MMP-1 and MMP-9 by activated monocytes. The central actin-binding domain, amino acids 17-23, had all of the activity for metalloproteinase induction. We conclude that part of the wound healing activity of Tbeta4 resides in its ability to increase proteinase activity via its central actin-binding domain. Thus, Tbeta4 may play a pivotal role in extracellular matrix remodeling during wound repair.     

miR-155 promotes cutaneous wound healing through enhanced keratinocytes migration by MMP-2

Inflammation, re-epithelization and tissue remodeling are three essential steps during wound healing. The re-epithelization process plays the most important role which mainly involves keratinocyte proliferation and migration. miR-155 has been reported to participate in cell migration and transformation, however, its function in skin wound healing is largely unknown. Here we hypothesize that overexpression of miR-155 at wound edges could accelerate wound healing mediated by enhanced keratinocyte migration. To test this hypothesis, direct local injection of miR-155 expression plasmid to wound edges was conducted to overexpress miR-155 in vivo. Results shown that miR-155 significantly promoted wound healing and re-epithelization compared to control, while did not affect wound contraction. Also, miR-155 overexpression accelerated primarily cultured keratinocyte migration in vitro, but had no effect on cell proliferation. Importantly, western blot analysis shown that MMP-2 was significantly upregulated whiles its inhibitor TIMP-1 downregulated after miR-155 treatment. Moreover, the use of ARP-101, an MMP-2 inhibitor, effectively attenuated the accelerative effects on cell migration induced by miR-155. Taken together, our results suggest that miR-155 has the promote effect on wound healing that is probably mediated by accelerating keratinocyte migration via upregulated MMP-2 level. This study provides a rationale for the therapeutic effect of miR-155 on wound healing.     

Matrix metalloproteinase 9 (MMP-9) is upregulated during scarless wound healing in athymic nude mice

Cutaneous wound healing is associated with migratory and remodeling events that require the action of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Differences in their expressions were observed during scar-forming and scar-free skin wound healing. We previously found that athymic nude mice are exceptional among mature mammals in their ability to heal injured skin scarlessly. The present study was undertaken to determine whether the modulation of MMP-2 and MMP-9 expression during scarless healing in nude mice was different from scar-forming animals. Full thickness skin wounds were made into the back of nude, wild-type controls (C57BL/6J), immunodeficient SCID and Rag, thymectomized neonates and adults, and cyclosporin A treated mice. Post-injured skin tissues were harvested at Day 7 and 24 after injury. Quantitative RT-PCR, Western blot, gelatin zymography and immunohistochemical assays were performed. Our results show that MMP-2 protein was high but similarly expressed in all post-injured animals on Day 7 after injury. Late phase (Day 24) of wound repair was characterized by a decrease in mRNA and protein expression and a decrease in gelatinolytic activity of MMP-2 in all post-injured samples. On the contrary, high (p < 0.001) levels of mRNA expression, prominent pro-and active forms of MMP-9 and cells immunopositive for MMP-9 were present exclusively in the post-injured tissues from nude mice on Day 24 after wounding. This data suggest that MMP-9 expression in the remodeling phase of wound healing in nude mice could be a major component of their ability for scar-free healing.     

Activation of MMP-9 by human lung epithelial cells in response to the cystic fibrosis-associated pathogen Burkholderia cenocepacia reduced wound healing in vitro

Burkholderia cepacia complex is a group of bacterial pathogens that cause opportunistic infections in cystic fibrosis (CF). The most virulent of these is Burkholderia cenocepacia. Matrix metalloproteinases (MMPs) are upregulated in CF patients. The aim of this work was to examine the role of MMPs in the pathogenesis of B. cepacia complex, which has not been explored to date. Real-time PCR analysis showed that B. cenocepacia infection upregulated MMP-2 and MMP-9 genes in the CF lung cell line CFBE41o- within 1 h, whereas MMP-2, -7, and -9 genes were upregulated in the non-CF lung cell line 16HBE14o-. Conditioned media from both cell lines showed increased MMP-9 activation following B. cenocepacia infection. Conditioned media from B. cenocepacia-infected cells significantly reduced the rate of wound healing in confluent lung epithelia (P < 0.05), in contrast to conditioned media from Pseudomonas aeruginosa-infected cells, which showed predominant MMP-2 activation. Treatment of control conditioned media from both cell lines with the MMP activator 4-aminophenylmercuric acetate (APMA) also resulted in clear activation of MMP-9 and to a much lesser extent MMP-2. APMA treatment of control media also delayed the repair of wound healing in confluent epithelial cells. Furthermore, specific inhibition of MMP-9 in medium from cells exposed to B. cenocepacia completely reversed the delay in wound repair. These data suggest that MMP-9 plays a role in the reduced epithelial repair observed in response to B. cenocepacia infection and that its activation following B. cenocepacia infection contributes to the pathogenesis of this virulent pathogen.     

Matrix metalloproteinases and the ontogeny of scarless repair: the other side of the wound healing balance

Early gestation mammalian fetuses possess the remarkable ability to heal cutaneous wounds in a scarless fashion. Over the past 20 years, scientists have been working to decipher the mechanisms underlying this phenomenon. Much of the research to date has focused on fetal correlates of adult wound healing that promote fibrosis and granulation tissue formation. It is important to remember, however, that wound repair consists of a balance between tissue synthesis, deposition, and degradation. Relatively little attention has been paid to this latter component of the fetal wound healing process.In this study, we examined the ontogeny of ten matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in nonwounded fetal rat skin and fibroblasts as a function of gestational age. We used a semiquantitative polymerase chain reaction protocol to analyze these important enzymes at time points that represent both the scarless and scar-forming periods of rat gestation. The enzymes evaluated were collagenase-1 (MMP-1), stromelysin-1 (MMP-3), gelatinase A (MMP-2), gelatinase B (MMP-9), membrane-type matrix metalloproteinases (MT-MMPs) 1, 2, and 3, and TIMPs 1, 2, and 3.Results demonstrated marked increases in gene expression for MMP-1, MMP-3 and MMP-9 that correlated with the onset of scar formation in nonwounded fetal skin. Similar results were noted in terms of MMP-9 gene expression in fetal fibroblasts. These results suggest that differences in the expression of these matrix metalloproteinases may have a role in the scarless wound healing phenotype observed early in fetal rat gestation. Furthermore, our data suggest that the differential expression of gelatinase B (MMP-9) may be mediated by the fetal fibroblasts themselves.     

MMP-3 provokes CTGF/CCN2 production independently of protease activity and dependently on dynamin-related endocytosis, which contributes to human dental pulp cell migration

Matrix metalloproteinase-3 (MMP-3) expression is promoted after pulpotomy, and application of MMP-3 to dental pulp after pulpotomy accelerates angiogenesis and hard tissue formation. However, the mechanism by which MMP-3 promotes dental pulp wound healing is still unclear. Connective tissue growth factor/CCN family 2 (CTGF/CCN2), a protein belonging to the CCN family, is considered to participate in wound healing, angiogenesis, and cell migration. In this study, we examined the involvement of CTGF/CCN2 in MMP-3-induced cell migration in human dental pulp (fibroblast-like) cells. In human dental pulp cells, MMP-3 promoted cell migration, but this effect was clearly blocked in the presence of anti-CTGF/CCN2 antibody. MMP-3 provoked mRNA and protein expression and secretion of CTGF/CCN2 in a concentration- and time-dependent manner. The MMP-3 inhibitor NNGH failed to suppress MMP-3-induced CTGF/CCN2 protein expression. The potent dynamin inhibitor dynasore clearly inhibited MMP-3-induced CTGF/CCN2 expression. These results strongly suggest that MMP-3 induces CTGF/CCN2 production independently of the protease activity of MMP-3 and dependently on dynamin-related endocytosis, which is involved in cell migration in human dental pulp cells.     

Reference values of concentrations of matrix metalloproteinases-1, -2, -9 and the tissue inhibitor of matrix metalloproteinases (TIMP-1) in the amniotic fluid during physiological pregnancy and delivery

The aim of this study was to determine reference values of matrix metalloproteinases (MMPs) MMP-1, MMP-2, MMP-9 and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) in the amniotic fluid at the first stage of labor in physiological pregnancy. Eighty nine women at the first stage of term labor have been examined. Samples of amniotic fluid were taken at the first period of labor by vaginal amniotomy. Concentrations of MMP-1, MMP-2, MMP-9, and TIMP-1 have been investigated in amniotic fluid samples by ELISA kits. The following normal concentration ranges for MMP-1, MMP-2, MMP-9, TIMP-1, and ratios of concentrations of MMPs and TIMP-1 (MMP-1/TIMP-1, MMP-2/TIMP-1, MMP-9/TIMP-1) have been determined for amniotic fluid samples obtained during the first period of labor in physiological pregnancy. These included: MMP-1: 5.1–16.8 pg/mg of protein; MMP-2: 238.3–374.1 pg/mg of protein; MMP-9: 66.1–113.3 pg/mg of protein, TIMP-1: 4.7–13.6 pg/mg of protein, MMP-1/TIMP-1 ratio: 0.1–2.2, MMP-2/TIMP-1 ratio: 19.9–55.7; MMP-9/TIMP-1 ratio: 4.2–17.2.     

Dedifferentiation-specific expression of MMP-9 and the effects of RA on its expression during salamander limb regeneration

The matrix metalloproteinases (MMPs) are well known to responsible for the degradation of extracellular matrix (ECM) during tissue remodelling such as wound healing, metamorphosis, and regeneration. In present study, gelatinase activities were investigated in normal and retinoic acid (RA)-treated limb regenerates. During the early phase of limb regeneration, gelatinase activities increased greatly, and RA caused the enhanced and prolonged gelatinase activities. We also isolated full length of Hynobius MMP-9, and its spatial and temporal expression profiles were examined in normal, RA-treated, and denervated limb regenerates. Whole mount in situ hybridization showed that the expression of MMP-9 increased in the wound epidermis at the wound healing stage and early phase of dedifferentiation stage. In addition, RA enhanced remarkably its expression both in terms of level and duration in the wound epidermis. However, expression signal of MMP-9 was barely detectable in denervated in limb regenerates. Our results may indicate that MMP-9 plays important role(s) in the dedifferentiation process by participating in ECM degradation and enhancement of MMP-9 expression and activity might be closely related to RA-evoked pattern duplication.     

Matrix metalloproteinase-8 regulates transforming growth factor-β1 levels in mouse tongue wounds and fibroblasts in vitro

Matrix metalloproteinase-8 (MMP-8)-deficient mice (Mmp8-/-) exhibit delayed dermal wound healing, but also partly contradicting results have been reported. Using the Mmp8-/- mice we investigated the role of MMP-8 in acute wound healing of the mobile tongue, and analyzed the function of tongue fibroblasts in vitro. Interestingly, in the early phase the tongue wounds of Mmp8-/- mice healed faster than those of wild type (wt) mice resulting in significant difference in wound widths (P=0.001, 6–24 h). The Mmp8-/- wounds showed no change in myeloperoxidase positive myeloid cell count, but the level of transforming growth factor (TGF)-β1 was significantly increased (P=0.007) compared to the wt tongues. Fibroblasts cultured from wt tongues expressed MMP-8 and TGF-β1. However, higher TGF-β1 levels were detected in Mmp8-/- fibroblasts, and MMP-8 treatment decreased phosphorylated Smad-2 levels and α-smooth muscle actin expression in these fibroblasts suggesting reduced TGF-β1 signaling. Consistently, a degradation of recombinant TGF-β1 by MMP-8 decreased its ability to activate the signaling cascade in fibroblasts. Moreover, collagen gels with Mmp8-/- fibroblasts reduced more in size. We conclude that MMP-8 regulates tongue wound contraction rate and TGF-β1 levels. In vitro analyses suggest that MMP-8 may also play a role in regulating TGF-β1 signaling of stromal fibroblasts.     

Fluid shear stress regulates metalloproteinase-1 and 2 in human periodontal ligament cells: Involvement of extracellular signal-regulated kinase (ERK) and P38 signaling pathways

Matrix metalloproteinase (MMP)-1, 2, with their endogenous inhibitors, tissue inhibitor of metalloproteinase (TIMP)-1, 2 are critical for extracellular matrix remodeling in human periodontal ligament (PDL) and their expression are sensitive to mechanical stresses. Shear stress as the main type of mechanical stress in tooth movement is involved in matrix turnover. However, how shear stress regulates MMPs and TIMPs system is still unclear. In this study, we investigated the effect of fluid shear stress on expression of MMP-1, 2 and TIMP-1, 2 in human PDL cells and the possible roles of mitogen-activated protein kinases in this process. Three levels of fluid shear stresses (6, 9 and 12dyn/cm(2)) were loaded on PDL cells for 2, 4, 8 and 12h. The results indicated that fluid shear stress rearranged cytoskeleton in PDL cells. Fluid shear stress increased expression of MMP-1, 2, TIMP-1 and suppressed TIMP-2 expression. MAP kinases including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 were activated rapidly by fluid shear stress. The ERK inhibitor blocked fluid shear stress induced MMP-1 expression and P38 inhibitor reduced fluid shear stress stimulated MMP-2 expression. Our study suggested that fluid shear stress involved in PDL remodeling via regulating MMP-1, 2 and TIMP-1, 2 expression. ERK regulated fluid shear stress induced MMP-1 expression and P38 play a role in fluid shear stress induced MMP-2 upregulation.