Changes in human burn fluid biological activity during normal burn healing

The main goal of this work was monitoring the changes occurring in human burn fluid biological activity during normal burn healing. The fluid available in the burn until healing makes a good material for controlling biochemical microenvironment of burn cells. This environment involves factors, such as extracellular matrix proteins and matrix metalloproteinases. In this work our previous studies of the influence of wound and burn fluids on the functional activity of cells were extended to include the effect of burn fluid on fibroblasts and keratinocytes, i. e. human skin cells present in the wound and involved in wound healing. It was shown that human burn fluid biological activity depends on the time that passed after burning, and on the correctness of healing. Migration of human fibroblasts becomes more intensive under the influence of such a fluid independently on the time of fluid sampling. Unlike, keratinocyte migration was inhibited by burn fluid sampled 1-3 days after burning but was enhanced by fluids sampled 6 days following burning. The obtained data are to be necessarily taken into consideration at burn treatment and also at transplantation of cells for healing of wounds of different nature.     

Cholesterol Sulfate Alters Substrate Preference of Matrix Metalloproteinase-7 and Promotes Degradations of Pericellular Laminin-332 and Fibronectin

Localization of secreted matrix metalloproteinases (MMPs) on the cell surface is required not only for processing of cell surface proteins, but also for controlled degradation of the extracellular matrix (ECM). Our previous study demonstrated that binding of MMP-7 (matrilysin) to cell surface cholesterol sulfate (CS) is essential for the cell membrane-associated proteolytic action of this MMP. In this study, we investigated the role of CS in the MMP-7-catalyzed degradation of protein components of ECM. We found that the degradation of laminin-332 (laminin-5) catalyzed by MMP-7 was accelerated dramatically in the presence of CS, whereas the sulfated lipid inhibited the degradation of casein catalyzed by the protease. The MMP-7-catalyzed degradation of fibronectin was partially inhibited in the presence of low concentrations of CS, whereas it was accelerated significantly at high concentrations of the lipid. Therefore, it is likely that CS alters the substrate preference of MMP-7. We also found that the proteins of which MMP-7-catalyzed degradation were accelerated by CS also had affinities for CS, suggesting that CS facilitates the proteolyses by cross-linking MMP-7 to its substrates. Moreover, MMP-7 tethered to cancer cell surface via CS degraded fibronectin and laminin-332 coated on a culture plate. The degradations of the adhesive proteins led to significant detachment of the cells from the plate. Taken together, our findings provide a novel mechanism in which cell surface CS promotes the proteolytic activities of MMP-7 toward selective substrates in the pericellular ECM, thereby contributing to cancer cell migration and metastasis.     

Matrix metalloproteinase-2 is involved in A549 cell migration on laminin-10/11

We have reported that laminin-10/11 strongly promotes migration of A549 human lung carcinoma cells by activating the alpha3beta1 integrin-dependent signaling pathway. To elucidate the mechanism involved, we investigated whether matrix metalloproteinases (MMPs) are involved in cell migration on laminin-10/11. Here, we demonstrate that laminin-10/11, but not fibronectin which does not greatly promote A549 cell movement, stimulated MMP-2 secretion approximately 3-fold. The cell migration-promoting activity of laminin-10/11 was down-regulated by an MMP inhibitor. In addition, cell motility was significantly increased when cells adhered to a mixture of fibronectin and laminin-10/11 with a concomitant decrease of focal contacts, compared with those adhering to fibronectin alone. The enhanced cell migration was partially suppressed by the MMP inhibitor. Furthermore, an anti-alpha3 integrin, but not an anti-alpha5 integrin, antibody induced the activated form of MMP-2. These data suggest that MMP-2 may play an important role in A549 cell migration on laminin-10/11 through an alpha3beta1 integrin-dependent pathway.     

Laminin-10/11 and fibronectin differentially prevent apoptosis induced by serum removal via phosphatidylinositol 3-kinase/Akt- and MEK1/ERK-dependent pathways

Cell adhesion to the extracellular matrix inhibits apoptosis, but the molecular mechanisms underlying the signals transduced by different matrix components are not well understood. Here, we examined integrin-mediated antiapoptotic signals from laminin-10/11 in comparison with those from fibronectin, the best characterized extracellular adhesive ligand. We found that the activation of protein kinase B/Akt in cells adhering to laminin-10/11 can rescue cell apoptosis induced by serum removal. Consistent with this, wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, or ectopic expression of a dominant-negative mutant of Akt selectively accelerated cell death upon serum removal. In contrast to laminin-10/11, fibronectin rescued cells from serum depletion-induced apoptosis mainly through the extracellular signal-regulated kinase pathway. Cell survival on fibronectin but not laminin was significantly reduced by treatment with PD98059, a specific inhibitor of mitogen- or extracellular signal-regulated kinase kinase-1 (MEK1) and by expression of a dominant-negative mutant of MEK1. Laminin-10/11 was more potent than fibronectin in preventing apoptosis induced by serum depletion. These results, taken together, demonstrate laminin-10/11 potency as a survival factor and demonstrate that different extracellular matrix components can transduce distinct survival signals through preferential activation of subsets of multiple integrin-mediated signaling pathways.     

Activities of the matrix metalloproteinase stromelysin-2 (MMP-10) in matrix degradation and keratinocyte organization in wounded skin

The matrix metalloproteinase stromelysin-2 is expressed in keratinocytes of the epithelial tongue of skin wounds, suggesting a role in keratinocyte migration. Here, we show that stromelysin-2 enhances migration of cultured keratinocytes. To gain insight into the in vivo activities of stromelysin-2 in epithelial repair, we generated transgenic mice expressing a constitutively active stromelysin-2 mutant in keratinocytes. These animals had no alterations in skin architecture, and the healing rate of skin wounds was normal. Histologically, however, we found abnormalities in the organization of the wound epithelium. Keratinocytes at the migrating epidermal tip were scattered in most sections of mice with high expression level, and there was a reduced deposition of new matrix. In particular, the staining pattern of laminin-5 at the wound site was altered. This may be due to proteolytic processing of laminin-5 by stromelysin-2, because degradation of laminin-5 by this enzyme was observed in vitro. The inappropriate matrix contact of keratinocytes was accompanied by aberrant localization of beta1-integrins and phosphorylated focal adhesion kinase, as well as by increased apoptosis of wound keratinocytes. These results suggest that a tightly regulated expression level of stromelysin-2 is required for limited matrix degradation at the wound site, thereby controlling keratinocyte migration.     

Human fibronectin and MMP-2 collagen binding domains compete for collagen binding sites and modify cellular activation of MMP-2.

The region of fibronectin (FN) surrounding the two type II modules of FN binds type I collagen. However, little is known about interactions of this collagen binding domain with other collagen types or extracellular matrix molecules. Among several expressed recombinant (r) human FN fragments from the collagen binding region of FN, only rI6-I7, which included the two type II modules and both flanking type I modules, bound any of several tested collagens. The rI6-I7 interacted specifically with both native and denatured forms of types I and III collagen as well as denatured types II, IV, V and X collagen with apparent K(d) values of 0.2-3.7 x 10(-7) M. Reduction with DTT disrupted the binding to gelatin verifying the functional requirement for intact disulfide bonds. The FN fragments showed a weak, but not physiologically important, binding to heparin, and did not bind elastin or laminin. The broad, but selective range of ligand interactions by rI6-I7 mirrored our prior observations for the collagen binding domain (rCBD) from matrix metalloproteinase-2 (MMP-2) [J. Biol. Chem. 270 (1995) 11555]. Subsequent experiments showed competition between rI6-I7 and rCBD for binding to gelatin indicating that their binding sites on this extracellular matrix molecule are identical or closely positioned. Two collagen binding domain fragments supported cell attachment by a beta1-integrin-dependent mechanism although neither protein contains an Arg-Gly-Asp recognition sequence. Furthermore, activation of MMP-2 and MMP-9 was greatly reduced for HT1080 fibrosarcoma cells cultured on either of the fibronectin fragments compared to full-length FN. These observations imply that the biological activities of FN in the extracellular matrix may involve interactions with a broad range of collagen types, and that exposure to pathologically-generated FN fragments may substantially alter cell behavior and regulation.     

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.     

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.     

Matrix metalloproteinase 2 (gelatinase A) is related to migration of keratinocytes.

The role of matrix metalloproteinases (MMPs) in cell migration was studied by measuring cell growth, migration, and production of MMP-2 and -9 in oral mucosal and skin keratinocytes cultured in the presence of synthetic MMP inhibitors. MMP-2 was the major gelatinolytic MMP produced by these cells while MMP-9 was produced at a low basal level. Inhibitor effects on MMP-9 production were therefore studied in keratinocytes stimulated by tumor necrosis factor alpha (TNFalpha). Tetracycline analogues at concentrations that inhibited the production of MMP-2 but not MMP-9 were able to drastically inhibit migration of both mucosal and skin keratinocytes. Tetracycline analogues also inhibited keratinocyte growth, an effect not found for the other inhibitors tested. Heterocyclic carbonate-derived compounds (LWs) that inhibited MMP-9 but not MMP-2 production had no effect on cell migration. Batimastat, a potent MMP inhibitor, did not have any effect on MMP production or cell growth but did inhibit keratinocyte migration. Tumor growth factor beta (TGFbeta) increased keratinocyte migration as well as both cell-associated and secreted MMP-2 production in wounded cell cultures. The secreted enzyme was partially converted into an active form. In this model batimastat totally blocked TGFbeta-promoted keratinocyte migration. Immunostaining of keratinocytes advancing into the wound revealed that MMP-2 was localized in extracellular matrix contactlike structures against the endogenously produced laminin-5-rich matrix. MMP-9 was localized diffusely along the cell membranes. Using in situ hybridization we observed that in chronically inflamed human gingiva MMP-2 is expressed in epithelium extending into subepithelial connective tissue. These results suggest that MMP-2 plays a specific role in epithelial migration, possibly by detaching the advancing cells from the pericellular matrix or by activating other MMPs.     

Crystal structure of an active form of human MMP-1

The extracellular matrix is a dynamic environment that constantly undergoes remodelling and degradation during vital physiological processes such as angiogenesis, wound healing, and development. Unbalanced extracellular matrix breakdown is associated with many diseases such as arthritis, cancer and fibrosis. Interstitial collagen is degraded by matrix metalloproteinases with collagenolytic activity by MMP-1, MMP-8 and MMP-13, collectively known as the collagenases. Matrix metalloproteinase 1 (MMP-1) plays a pivotal role in degradation of interstitial collagen types I, II, and III. Here, we report the crystal structure of the active form of human MMP-1 at 2.67 A resolution. This is the first MMP-1 structure that is free of inhibitor and a water molecule essential for peptide hydrolysis is observed coordinated with the active site zinc. Comparing this structure with the human proMMP-1 shows significant structural differences, mainly in the relative orientation of the hemopexin domain, between the pro form and active form of the human enzyme.     

Keratinocyte-derived Laminin-332 Protein Promotes Melanin Synthesis via Regulation of Tyrosine Uptake

Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake.     

MMP-2 inhibition reduces renal macrophage infiltration with increased fibrosis in UUO

We examined the role of matrix metalloproteinase-2 (MMP-2) in renal fibrosis and its effect on interstitial macrophage infiltration in a mouse model of unilateral ureteral obstruction (UUO). TISAM, a selective inhibitor of MMP-2, was administered during early stage (day -2 to 4; protocol A) and late stage (day 7 to 13; protocol B) after UUO. Treatment with TISAM accelerated fibrosis both at day 5 (A) and at day 14 (B). The degree of macrophage infiltration was decreased by the treatment with TISAM at day 14, but not at day 5. In vitro macrophage migration assay showed a greater migration to renal tissue of control UUO kidney (day 14) than to TISAM-treated kidney, which was suppressed by preincubating macrophages with RGDS, a fibronectin degradation peptide. These results suggest that MMP-2 acts to accelerate macrophage infiltration in the late stage of UUO, possibly by degrading extracellular matrix components.     

Dermatopontin interacts with fibronectin, promotes fibronectin fibril formation, and enhances cell adhesion

We report that dermatopontin (DP), an abundant dermal extracellular matrix protein, is found in the fibrin clot and in the wound fluid, which comprise the provisional matrix at the initial stage of wound healing. DP was also found in the serum but at a lower concentration than that in wound fluid. DP co-localized with both fibrin and fibronectin on fibrin fibers and interacted with both proteins. Both normal fibroblast and HT1080 cell adhesion to the fibrin-fibronectin matrix were dose-dependently enhanced by DP, and the adhesion was mediated by α5β1 integrin. The cytoskeleton was more organized in the cells that adhered to the fibrin-fibronectin-DP complex. When incubated with DP, fibronectin formed an insoluble complex of fibronectin fibrils as visualized by electron microscopy. The interacting sites of fibronectin with DP were the first, thirteenth, and fourteenth type III repeats (III(1), III(13), and III(14)), with III(13) and III(14) assumed to be the major sites. The interaction between III(2-3) and III(12-14) was inhibited by DP, whereas the interaction between I(1-5) and III(12-14) was specifically and strongly enhanced by DP. Because the interaction between III(2-3) and III(12-14) is involved in forming a globular conformation of fibronectin, and that between I(1-5) and III(12-14) is required for forming fibronectin fibrils, DP promotes fibronectin fibril formation probably by changing the fibronectin conformation. These results suggest that DP has an accelerating role in fibroblast cell adhesion to the provisional matrix in the initial stage of wound healing.     

In vivo localization of gelatinases (MMP-2 and -9) by in situ zymography with a selective gelatinase inhibitor

In situ zymography provides a tool to localize proteolytic activity in tissues in vivo. However, it has been difficult to discriminate between the proteases responsible for the detected activity. We used a selective tissue-permeable gelatinase inhibitor, the CTTHWGFTLC-peptide (CTT) in inflamed human gingiva. The CTT-peptide was evidenced to home, target to, and selectively inhibit the areas of gelatinolytic activity in inflamed human gingiva expressing MMP-2 and -9. Gelatinolytic activity, MMP-9 immunoreactivity, and mRNA expression as well as CD-45-positive inflammatory cells colocalized well in the inflamed human gingival connective tissue. Gelatinolytic activity corresponding to MMP-2 colocalized with laminin-5 gamma2-chain immunoreactivity and was detected in the close vicinity of the sulcular basement membrane region. Furthermore, the CTT-peptide inhibited beta-caseinolysis by human MMP-2 and MMP-9 as well as laminin-5 gamma2-chain degradation by MMP-2 in vitro. Thus, the CTT-peptide may prove to be a useful tool (i) to discriminate between gelatinolytic proteases detected by in situ zymography and (ii) to preventMMP-2-dependent induction of epithelial cell migration and gelatinase-dependent tissue destruction in inflammatory and malignant diseases.     

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.     

Selective modulation of matrix metalloproteinase 9 (MMP-9) functions via exosite inhibition

Unregulated activities of the matrix metalloproteinase (MMP) family have been implicated in primary and metastatic tumor growth, angiogenesis, and pathological degradation of extracellular matrix components, such as collagen and laminin. However, clinical trials with small molecule MMP inhibitors have been largely unsuccessful, with a lack of selectivity considered particularly problematic. Enhanced selectivity could be achieved by taking advantage of differences in substrate secondary binding sites (exosites) within the MMP family. In this study, triple-helical substrates and triple-helical transition state analog inhibitors have been utilized to dissect the roles of potential exosites in MMP-9 collagenolytic behavior. Substrate and inhibitor sequences were based on either the alpha1(V)436-450 collagen region, which is hydrolyzed at the Gly (downward arrow) Val bond selectively by MMP-2 and MMP-9, or the Gly (downward arrow) Leu cleavage site within the consensus interstitial collagen sequence alpha1(I-III)769-783, which is hydrolyzed by MMP-1, MMP-2, MMP-8, MMP-9, MMP-13, and MT1-MMP. Exosites within the MMP-9 fibronectin II inserts were found to be critical for interactions with type V collagen model substrates and inhibitors and to participate in interactions with an interstitial (types I-III) collagen model inhibitor. A triple-helical peptide incorporating a fibronectin II insert-binding sequence was constructed and found to selectively inhibit MMP-9 type V collagen-based activities compared with interstitial collagen-based activities. This represents the first example of differential inhibition of collagenolytic activities and was achieved via an exosite-binding triple-helical peptide.     

Isolation of human basal keratinocytes by selective adhesion to extracellular matrix proteins

Epidermal human cells (keratinocytes) differently interact with extracellular matrix proteins of the skin basal membrane depending on the stages of their differentiation. The pool of basal keratinocytes commonly includes stem cells and transient amplifying cells. They directly attach to the skin basal membrane. Keratinocytes change their adhesive properties during differentiation, lose direct interaction with the basal membrane and move to suprabasal epidermal strata. From this, it is suggested that basal and primarily stem cells can be isolated from a heterogenous keratinocyte population due to their selective adhesion to the extracellular matrix proteins. In the current study, we analysed the specificity of interaction between primary keratinocytes and extracellular matrix proteins (collagens of I and IV types, laminin-2/4, fibronectin and matrigel). We have demonstrated that the basal keratinocytes extracted from the skin have different adhesive abilities. The rapidly spreading cells usually interacted with collagen and fibronectin rather that with laminin-2/4 or matrigel. The majority of these cells being represented by basal keratinocytes. Our data demonstrate that the applied method of keratinocyte selection may be directed for precise isolation of skin stem from a common cell population.     

Targeting of cytoskeletal linker proteins to focal adhesion complexes is reduced in fibroblasts adhering to laminin-1 when compared to fibronectin

Cellular interactions with the extracellular matrix determine to a large extent cell behavior, including cell migration. These interactions take place at specialized cellular structures, the focal adhesions, which have a substrate-specific morphology. To determine the molecular and functional relevance of this observation, the composition of isolated focal adhesions developed by fibroblasts adhering to fibronectin or laminin-1 was analyzed by indirect immunofluorescence and immunoblotting with or without stabilization of the structures by cross-linking. In the absence of cross-linking, integrins, talin, vinculin and, to a lower extent, paxillin remained associated with the focal adhesions formed on both substrates, indicating a tight association of these proteins with the extracellular matrix support. By contrast, alpha-actinin, FAK, and actin were apparently loosely maintained within focal adhesions and were found associated to these structures only after stabilization by cross-linking. Interestingly, although both substrates induced clustering and aggregation of all these proteins, their relative concentration, with the exception of alpha-actinin, was lower within the focal adhesions formed on laminin-1 than in those formed on fibronectin. Moreover, as assessed in migration assays, the locomotory speed of fibroblasts was higher on laminin-1 than on fibronectin. Altogether these results indicate that integrins involved in cellular interactions with fibronectin or laminin-1 trigger the formation of focal adhesion structures which differ by molecular organization, concentration in several adhesion plaque components, and function.     

MMP-2及MMP-9与主动脉疾病相关性的研究进展

基质金属蛋白酶是一类可降解细胞外基质的蛋白酶,基质金属蛋白酶-2和-9为明胶酶,可降解细胞外基质中的胶原蛋白及弹性蛋白,其动态平衡对维持细胞外基质的稳定具有重要意义。主动脉的细胞外基质是主动脉中层重要的组成部分,细胞外基质成分的改变可导致主动脉中层结构的损伤,在主动脉疾病的发生、发展过程中起着重要作用。主动脉基质金属蛋白酶-2和-9的表达失衡可引起主动脉中层细胞外基质的降解,导致主动脉中层结构的损伤,从而促进主动脉疾病的发生。同时,主动脉疾病也可导致血浆中MMP-2、MMP-9浓度的升高。本文对近年来基质金属蛋白酶与主动脉疾病相关性的研究及进展作一综述,为心血管疾病发生机制的研究和治疗提供文献依据。