Rac1 mediates type I collagen-dependent MMP-2 activation. role in cell invasion across collagen barrier

Cell migration and proteolysis are two essential processes during tumor invasion and metastasis. Matrix metalloproteinase (MMP)-2 (type IV collagenase; gelatinase A), is implicated in tumor metastasis as well as in primary tumor growth. The Rho family of small GTPases regulates the dynamics of actin cytoskeleton associated with cell motility. In this report, we provide evidence that Rac1, one member of Rho-related small GTPases, is a mediator of MMP-2 activation in HT1080 fibrosarcoma cells cultured in three-dimensional collagen gel (3D-col) and that MMP-2 activation is required for Rac1-promoted cell invasion through collagen barrier. Stable expression of dominant negative (Rac1V12N17) and constitutively active Rac1 (Rac1V12), respectively, in HT1080 cells demonstrates that Rac1 promoted cell invasiveness across type I collagen and collagen-dependent MMP-2 activation. Active Rac1 is sufficient to induce MMP-2 activation in cells cultured in fibrin gel, an extracellular matrix component that does not support MMP-2 activation. The Rac1-dependent MMP-2 activation occurred in a cell-associated fashion and required MMP activities. Because the cell membrane-mediated MMP-2 activation requires MT1-MMP and low amount of issue inhibitor of matrix metalloproteinase-2 (TIMP-2), their expression was examined. Rac1 modulated MT1-MMP mRNA level and the accumulation of a 43-kDa form of MT1-MMP protein, in correlation with MMP-2 activation profile. However, TIMP-2 expression was independent of Rac1 activity. The coordinate modulation of MMP-2 activity and MT1-MMP expression/processing by Rac1 is consistent with cell collagenolytic activity. The C-terminal hemopexin-like domain of MMP-2, which interferes with the cell membrane activation of MMP-2, reduced Rac1-promoted cell invasiveness as monitored by collagen invasion assay. These results suggest that collagen-dependent MMP-2 activation and MT1-MMP expression/processing contribute to Rac-promoted tumor cell invasion through interstitial collagen barrier.     

Endothelin-1 is a potent stimulator of alpha1beta1 integrin-mediated collagen matrix remodeling by rat mesangial cells

Endothelin-1 (ET) is known to stimulate mesangial cell (MC) proliferation, extracellular matrix (ECM) synthesis, and thereby contribute to the progression of glomerulonephritis (GN). To clarify the molecular and cellular mechanisms of how ET is involved in the development of glomerular sclerosis, we investigated the influence of ET on the MC-alpha1beta1 integrin-mediated collagen matrix reorganization using a collagen gel contraction assay. ET enhanced MC-alpha1beta1 integrin-mediated gel contraction in a dose-dependent manner. Addition of the endothelin A (ETA) receptor antagonist, BQ123, into collagen gels abolished ET-induced gel contraction by MC. Cell behavior involved in ET-induced gel contraction was investigated in combination with function-blocking anti-alpha1-integrin antibody. Migration and adhesion assays revealed that ET stimulated alpha1beta1 integrin-mediated MC migration but did not influence cell adhesion to type I collagen (collagen I). Integrin-function blocking studies using anti-alpha1 integrin antibody indicated that MC-alpha1beta1 integrin is required not only for collagen-dependent migration, but also for gel contraction. Zymography showed that ET increased MC matrix metalloproteinase-2 (MMP-2) activity in a dose-dependent manner during MC-induced gel contraction process. Finally, flow cytometry analysis indicated that ET did not affect the cell surface expression of the MC-alpha1beta1 integrin within the collagen gel. These data suggested that ET promotes collagen matrix reorganization through the enhancement of MC-alpha1beta1 integrin-dependent migration and MMP-2 activity. We therefore conclude that ET is a potential molecule inducing pathological collagen matrix remodeling observed in progressive GN.     

The behaviour of BHK cells and neutrophil leukocytes on collagen gels of defined mechanical strength

This study demonstrates how the mechanical strength of a series of collagen/composite gels can be measured using a penetrometer. It was found that the presence of fibrin in collagen gels resulted in increased gel strength. Similarly hyaluronic acid was found to increase the strength of collagen gels. Addition of heparin weakened collagen gels as did chondroitin-6-sulphate. Neutrophil migration into collagen gels was found to be inversely proportional to gel strength. Fibrin and hyaluronic acid containing gels inhibited neutrophil migration while the presence of heparin and chondroitin sulphate increased neutrophil migration. BHK gel contraction experiments demonstrated how the presence of fibrin prevents gel contraction. Despite increasing gel strength the presence of hyaluronic acid appeared to have no effect on BHK contraction of collagen gels. Similarly the presence of heparin or chondroitin sulphate had no effect on gel contraction by BHK cells.     

Type VIII collagen signals via β1 integrin and RhoA to regulate MMP-2 expression and smooth muscle cell migration

The extracellular matrix signals and regulates the behavior of vascular cells during the pathogenesis of atherosclerosis. Type VIII collagen, a short chain collagen, is scarcely present in normal arteries, but is dramatically upregulated in atherosclerosis and after other types of vascular injury. Cell culture studies have revealed that this protein supports smooth muscle cell (SMC) adhesion and stimulates migration, however little is known about the signaling or the mechanisms by which this occurs. SMCs isolated from wild-type C57BL/6 and type VIII collagen deficient mice were studied using assays to measure chemotactic and haptotactic migration, and remodeling and contraction of 3-dimensional type I collagen gels. Col8^?/? SMCs exhibited impairments in migration, and a strongly adhesive phenotype with prominent stress fibers, stable microtubules and pronounced central basal focal adhesions. The addition of exogenous type VIII collagen to the Col8^?/? SMCs rescued the impairments in migration, and restored cytoskeletal architecture so that it was similar to Col8^+/+ cells. We measured elevated levels of active GTP-RhoA in the Col8^?/? cells, and this too was reversed by treatment with exogenous type VIII collagen. We showed that type VIII collagen normally suppresses RhoA activation through a beta-1 integrin dependent mechanism. MMP-2 levels were reduced in the Col8^?/? SMCs, and knockdown of MMP-2 in Col8^+/+ SMCs partially recapitulated the decreases in migration and 3D gel contraction seen in Col8^?/? cells, showing that type VIII collagen-stimulated migration was dependent on MMP-2. Inhibition of Rho restored MMP-2 activity in the Col8^?/? cells, and partially rescued migration, demonstrating that the elevations in RhoA activity were responsible for the suppression of migration of these cells. In conclusion, we have shown that type VIII collagen signals through beta-1 integrin receptors to suppress RhoA, allowing optimal configuration of the cytoskeleton, and the stimulation of MMP-2-dependent cell migration.     

The mechanical and pharmacological regulation of glioblastoma cell migration in 3D matrices

The invasion of glioblastoma is a complex process based on the interactions of tumor cells and the extracellular matrix. Tumors that are engineered using biomaterials are more physiologically relevant than a two-dimensional (2D) cell culture system. Matrix metalloproteinases and the plasminogen activator generated by tumor cells regulate a tumor’s invasive behavior. In this study, microtumors were fabricated by encapsulating U87 glioma cells in Type I collagen and then glioma cell migration in the collagen hydrogels was investigated. Crosslinking of collagen with 8S-StarPEG increased the hydrogel viscosity and reduced the tumor cell migration speed in the hydrogels. The higher migration speed corresponded to the increased gene expression of MMP-2, MMP-9, urokinase plasminogen activator (uPA), and tissue plasminogen activator (tPA) in glioma cells grown in non-crosslinked collagen hydrogels. Inhibitors of these molecules hindered U87 and A172 cell migration in collagen hydrogels. Aprotinin and tranexamic acid did not inhibit U87 and A172 migration on the culture dish. This study demonstrated the differential effect of pharmacologic molecules on tumor cell motility in either a 2D or three-dimensional culture environment.     

Dynamics of matrix metalloproteinases activity of primary murine embryonic fibroblasts during cultivation

Embryonic cells regulate the expression of matrix metalloproteinases (MMP) providing remodulation of extracellular matrix, which in turn provides the changes in cell adhesion and migration during the cell development and differentiation. In present work we studied the changes of gelatinases (MMP-2 and MMP-9) and collagenases (MMP-1 and MMP-8) activities in the process of cultivating the primary murine embryonic fibroblasts (MEF). Cultivation was continued for 6 passages, after that the culture died in time. According to gelatin and collagen zymography results, drastic changes of all MMPs activities occurred during the third passage of cell cultivation. The MMP-1 and MMP-9 activity appears in the middle of cultivation and then disappeared at the end. The most important event MEF cultivation is appearance and subsequent reservation of collagenase MMP-8 and active form of gelatinase MMP-2.     

The Activity of Collagenase-1 Is Required for Keratinocyte Migration on a Type I Collagen Matrix

We have shown in a variety of human wounds that collagenase-1 (MMP-1), a matrix metalloproteinase that cleaves fibrillar type I collagen, is invariably expressed by basal keratinocytes migrating across the dermal matrix. Furthermore, we have demonstrated that MMP-1 expression is induced in primary keratinocytes by contact with native type I collagen and not by basement membrane proteins or by other components of the dermal or provisional (wound) matrix. Based on these observations, we hypothesized that the catalytic activity of MMP-1 is necessary for keratinocyte migration on type I collagen. To test this idea, we assessed keratinocyte motility on type I collagen using colony dispersion and colloidal gold migration assays. In both assays, primary human keratinocytes migrated efficiently on collagen. The specificity of MMP-1 in promoting cell movement was demonstrated in four distinct experiments. One, keratinocyte migration was completely blocked by peptide hydroxymates, which are potent inhibitors of the catalytic activity of MMPs. Two, HaCaTs, a line of human keratinocytes that do not express MMP-1 in response to collagen, did not migrate on a type I collagen matrix but moved efficiently on denatured type I collagen (gelatin). EGF, which induces MMP-I production by HaCaT cells, resulted in the ability of these cells to migrate across a type I collagen matrix. Three, keratinocytes did not migrate on mutant type I collagen lacking the collagenase cleavage site, even though this substrate induced MMP-1 expression. Four, cell migration on collagen was completely blocked by recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1) and by affinity-purified anti–MMP-1 antiserum. In addition, the collagen-mediated induction of collagenase-1 and migration of primary keratinocytes on collagen was blocked by antibodies against the α2 integrin subunit but not by antibodies against the α1 or α3 subunits. We propose that interaction of the α2β1 integrin with dermal collagen mediates induction of collagenase-1 in keratinocytes at the onset of healing and that the activity of collagenase-1 is needed to initiate cell movement. Furthermore, we propose that cleavage of dermal collagen provides keratinocytes with a mechanism to maintain their directionality during reepithelialization.     

Fluid shear stress regulates the invasive potential of glioma cells via modulation of migratory activity and matrix metalloproteinase expression

Background

Glioma cells are exposed to elevated interstitial fluid flow during the onset of angiogenesis, at the tumor periphery while invading normal parenchyma, within white matter tracts, and during vascular normalization therapy. Glioma cell lines that have been exposed to fluid flow forces in vivo have much lower invasive potentials than in vitro cell motility assays without flow would indicate.

Methodology/Principal Findings

A 3D Modified Boyden chamber (Darcy flow through collagen/cell suspension) model was designed to mimic the fluid dynamic microenvironment to study the effects of fluid shear stress on the migratory activity of glioma cells. Novel methods for gel compaction and isolation of chemotactic migration from flow stimulation were utilized for three glioma cell lines: U87, CNS-1, and U251. All physiologic levels of fluid shear stress suppressed the migratory activity of U87 and CNS-1 cell lines. U251 motility remained unaltered within the 3D interstitial flow model. Matrix Metalloproteinase (MMP) inhibition experiments and assays demonstrated that the glioma cells depended on MMP activity to invade, and suppression in motility correlated with downregulation of MMP-1 and MMP-2 levels. This was confirmed by RT-PCR and with the aid of MMP-1 and MMP-2 shRNA constructs.

Conclusions/Significance

Fluid shear stress in the tumor microenvironment may explain reduced glioma invasion through modulation of cell motility and MMP levels. The flow-induced migration trends were consistent with reported invasive potentials of implanted gliomas. The models developed for this study imply that flow-modulated motility involves mechanotransduction of fluid shear stress affecting MMP activation and expression. These models should be useful for the continued study of interstitial flow effects on processes that affect tumor progression.     

Potential synergies between matrix proteins and soluble factors on resorption and proteinase activities of rabbit bone cells

Human growth hormone (GH) has recently been found to stimulate osteoclastic resorption, cysteine-proteinase and metalloproteinase activities (MMP-2 and MMP-9) in vitro via insulin-like growth factor-I (IGF-I) produced by stromal cells. The present study investigated the effects of two extracellular matrix components (vitronectin and type-I collagen) on hGH- and hIGF-1-stimulated osteoclastic resorption and proteinase activities in a rabbit bone cell model. After 4 days of rabbit bone cell culture on dentin slices with vitronectin coating, hGH and hIGF-1 stimulated bone resorption and hIGF-1 upmodulated cysteine-proteinase activities. MMP-2 expression (but not resorption, cathepsin or MMP-9 activities) was upmodulated by hGH and hIGF-1 on dentin slices coated with type I collagen as compared to those without coating. Then, vitronectin was synergistic with hIGF-1 in the regulation of cysteine-proteinase production whereas collagen showed synergy with hGH and hIGF-1 in the regulation of MMP-2 production. Anti-alphavbeta3 totally abolished the effects of hGH and hIGF-1 on metalloproteinase release, but had no influence on cathepsin release. The results suggest that cysteine-proteinase modulation is not mediated by alphavbeta3 integrin (strongly expressed on osteoclastic surface) whereas the resorption process and metalloproteinase modulation are clearly mediated by this integrin. Our finding about the collagen coating also suggests that hGH- and hIGF-1-stimulated MMP-2 activity are mediated, along with alphavbeta3 integrin, by another adhesion molecule.     

The type I collagen induction of MT1-MMP-mediated MMP-2 activation is repressed by alphaVbeta3 integrin in human breast cancer cells.

The influence of alphaVbeta3 integrin on MT1-MMP functionality was studied in human breast cancer cells of differing beta3 integrin status. Overexpression of beta3 integrin caused increased cell surface expression of alphaV integrin and increased cellular adhesion to extracellular matrix (ECM) substrates in BT-549, MDA-MB-231 and MCF-7 cells. beta3 integrin expression also enhanced the migration of breast cancer cells on ECM substrates and enhanced collagen gel contraction. In vivo, alphaVbeta3 cooperated with MT1-MMP to increase the growth of MCF-7 cells after orthotopic inoculation in immunocompromised mice, but had no influence on in vitro proliferation. Despite these stimulatory effects, overexpression of beta3 integrin suppressed the type I collagen (Col I) induced MMP-2 activation in all breast cancer cell lines analyzed. This was also evident in extracts from the MCF-7 tumors in vivo, where MMP-2 activation was stimulated by MT1-MMP transfection, but attenuated with beta3 integrin expression. Although our studies confirm important biological effects of alphaVbeta3 integrin on enhancing cell adhesion and migration, ECM remodeling and tumor growth, beta3 integrin caused reduced MMP-2 activation in response to Col I in vitro, which appears to be physiologically relevant, as it was also seen in tumor xenografts in vivo. The reduction of MMP-2 activation (and thus MT1-MMP activity) by alphaVbeta3 in response to Col I may be important in scenarios where cells which are activated for matrix degradation need to preserve some pericellular collagen, perhaps as a substrate for cell adhesion and migration, thus maintaining a balanced level of proteolysis required for efficient tumor growth.     

TGF-β1 facilitates MT1-MMP-mediated proMMP-9 activation and invasion in oral squamous cell carcinoma cells

Matrix metalloproteinase (MMP)-2 and MMP-9, also known as gelatinases or type IV collagenases, are recognized as major contributors to the proteolytic degradation of extracellular matrix during tumor invasion. Latent MMP-2 (proMMP-2) is activated by membrane type 1 MMP (MT1-MMP) on the cell surface of tumor cells. We previously reported that cell-bound proMMP-9 is activated by the MT1-MMP/MMP-2 axis in HT1080 cells treated with concanavalin A in the presence of exogenous proMMP-2. However, the regulatory mechanism of proMMP-9 activation remains largely unknown. Transforming growth factor (TGF)-β1 is frequently overexpressed in tumor tissues and is associated with tumor aggressiveness and poor prognosis. In this study, we examined the role of TGF-β1 on MT1-MMP-mediated proMMP-9 activation using human oral squamous cell carcinoma cells. TGF-β1 significantly increased the expression of MMP-9. By adding exogenous proMMP-2, TGF-β1-induced proMMP-9 was activated during collagen gel culture, which was suppressed by the inhibition of TGF-β1 signaling or MT1-MMP activity. This MT1-MMP-mediated proMMP-9 activation was needed to facilitate TGF-β1-induced cell invasion into collagen gel. Thus, TGF-β1 may facilitate MT1-MMP-mediated MMP-9 activation and thereby stimulate invasion of tumor cells in collaboration with MT1-MMP and MMP-2.     

Higher expression and activity of metalloproteinases in human cervical carcinoma cell lines is associated with HPV presence.

Matrix metalloproteinases (MMPs) MMP-2, MMP-9, and MT1-MMP are required for basement membrane degradation in cervical carcinoma. We evaluated the expression and activity of MMPs and their inhibitors RECK and TIMP-2 in 3 human invasive cervical carcinoma cell lines. Two HPV16-positive cell lines (SiHa and CaSki) and an HPV-negative cell line (C33A) were cultured either onto a type-I collagen gel, Matrigel, or plastic, to recreate their three-dimensional growth environment and evaluate the expression of these genes using quantitative real-time PCR. We also analyzed the gelatinolytic activity of MMP-2 and MMP-9 by zymography. We found that HPV (human papillomavirus)-positive cell lines express higher levels of MMP-2, MT1-MMP, and TIMP-2 than the HPV negative cell line. In addition, MMP-9 was expressed at very low levels in both HPV-negative and HPV-positive cell lines. We also observed that the expression of the RECK gene is higher in CaSki cells, being associated with higher pro-MMP-2 activity. Furthermore, Matrigel substrate influences MMP-2 expression in both SiHa and CaSki cells. On the other hand, we found that type-I collagen gel, but not Matrigel, can enhance pro-MMP-2 activity in all cell lines. Our results suggest that the presence of HPV is related to increased expression of MMP-2, MT1-MMP, and TIMP-2, and that pro-MMP-2 activity is higher in HPV-positive than in HPV-negative cells.     

Cumulative influence of matrix metalloproteinase-1 and -2 in the migration of melanoma cells within three-dimensional type I collagen lattices

During melanoma progression, migrating cells must cross human dermis, a type I collagen-rich tissue. We have show that MMP-1 and MMP-2 act in a cumulative manner in the in vitro invasion of a three-dimensional type I collagen matrix by melanoma cells. Two melanoma cell lines (M1Dor and M3Da) previously reported to secrete proMMP-2 in a direct relationship with their tumorigenic potential into nude mice were used (F. Capon et al., 1999, Clin. Exp. Metastasis 17, 463-469). The highly tumorigenic cell line (M3Da) displayed a five-fold faster migration rate in type I collagen matrix, compared to its lower tumorigenic counterpart (M1Dor). In parallel, activation of proMMP-2 was evidenced in M3Da- but not M1Dor-populated collagen lattices. Such enzyme activation was associated with a significant decrease in TIMP-2 and TIMP-1 production. Agents known to interfere with proMMP-2 activation, i.e., excess TIMP-2, furin convertase inhibitor, and alphavbeta3 blocking antibody, reduced by 30-40% the type I collagen invasive capacity of M3Da cells. By comparison, batimastat, a wide-spectrum MMP inhibitor, exhibited a more pronounced inhibitory effect (>70%). It suggested that other collagenases than MMP-2 could participate in type I collagen invasion. Collagenase-3 (MMP-13) was produced at low levels by melanoma cells whatever the cell culture conditions. In contrast, M3Da and M1Dor cells secreted collagenase-1 (MMP-1) following 48 h of culture on plastic dishes. Growing melanoma cells in type I collagen gel did not modify enzyme production, but induced proMMP-1 activation in M3Da but not M1Dor cell-populated lattices. Blocking the plasmin-mediated proMMP-1 activation by aprotinin inhibited type I collagen gel invasion by 30%. Since the combination of aprotinin and furin convertase inhibitor reduced collagen invasiveness by melanoma cells to a level comparable to that attained with batimastat, we conclude that both MMP-2 and MMP-1 are involved in such tissue invasion.     

Direct visualization of protease activity on cells migrating in three-dimensions

Determining the specific role(s) of proteases in cell migration and invasion will require high-resolution imaging of sites of protease activity during live-cell migration through extracellular matrices. We have designed a novel fluorescent biosensor to detect localized extracellular sites of protease activity and to test requirements for matrix metalloprotease (MMP) function as cells migrate and invade three-dimensional collagen matrices. This probe fluoresces after cleavage of a peptide site present in interstitial collagen by a variety of proteases including MMP-2, -9, and -14 (MT1-MMP) without requiring transfection or modification of the cells being characterized. Using matrices derivatized with this biosensor, we show that protease activity is localized at the polarized leading edge of migrating tumor cells rather than further back on the cell body. This protease activity is essential for cell migration in native cross-linked but not pepsin-treated collagen matrices. The new type of high-resolution probe described in this study provides site-specific reporting of protease activity and insights into mechanisms by which cells migrate through extracellular matrices; it also helps to clarify discrepancies between previous studies regarding the contributions of proteases to metastasis.     

Integrin alpha1beta1 mediates collagen induction of MMP-13 expression in MC615 chondrocytes

During endochondral ossification, type I collagen is synthesized by osteoblasts together with some hypertrophic chondrocytes. Type I collagen has also been reported to be progressively synthesized in degenerative joints. Because Matrix Metalloproteinase-13 (MMP-13) plays an active role in remodeling cartilage in fetal development and osteoarthritic cartilage, we investigated whether type I collagen could activate MMP-13 expression in chondrocytes. We used a well-established chondrocytic cell line (MC615) and we found that MMP-13 expression was induced in MC615 cells cultured in type I collagen gel. We also found that alpha1beta1 integrin, a major collagen receptor, was expressed by MC615 cells and we further assessed the role of alpha1beta1 integrin in conducting MMP-13 expression. Induction of MMP-13 expression by collagen was potently and synergistically inhibited by blocking antibodies against alpha1 and beta1 integrin subunits, indicating that alpha1beta1 integrin mediates the MMP-13-inducing cellular signal generated by three-dimensional type I collagen. We also determined that activities of tyrosine kinase and ERK and JNK MAP kinases were required for this collagen-induced MMP-13 expression. Interestingly, bone morphogenetic protein (BMP)-2 opposed this induction, an effect that may be related to a role of BMP-2 in the maintenance of cartilage matrix.     

Matrix metalloproteinases 9 and 2 are necessary for the migration of Langerhans cells and dermal dendritic cells from human and murine skin

Dendritic cells migrate from the skin to the draining lymph nodes. They transport immunogenic MHC-peptide complexes, present them to Ag-specific T cells in the T areas, and thus generate immunity. Migrating dendritic cells encounter physical obstacles, such as basement membranes and collagen meshwork. Prior work has revealed that matrix metalloproteinase-9 (MMP-9) contributes to mouse Langerhans cell migration. In this study, we use mouse and human skin explant culture models to further study the role of MMPs in the migration and maturation of skin dendritic cells. We found that MMP-2 and MMP-9 are expressed on the surface of dendritic cells from the skin, but not from other sources. They are also expressed in migrating Langerhans cells in situ. The migration of both Langerhans cells and dermal dendritic cells is inhibited by a broad spectrum inhibitor of MMPs (BB-3103), by Abs to MMP-9 and -2, and by the natural tissue inhibitors of metalloproteinases (TIMP), TIMP-1 and TIMP-2. Inhibition by anti-MMP-2 and TIMP-2 define a functional role for MMP-2 in addition to the previously described function of MMP-9. The importance of MMP-9 was emphasized using MMP-9-deficient mice in which Langerhans cell migration from skin explants was strikingly reduced. However, MMP-9 was only required for Langerhans cell migration and not maturation, since nonmigrating Langerhans cells isolated from the epidermis matured normally with regard to morphology, phenotype, and T cell stimulatory function. These data underscore the importance of MMPs, and they may be of relevance for therapeutically regulating dendritic cell migration in clinical vaccination approaches.     

Macrophage migration in fibrin gel matrices. II. Effects of clotting factor XIII, fibronectin, and glycosaminoglycan content on cell migration

We investigated the migration of oil-induced, guinea pig peritoneal macrophages in three-dimensional fibrin matrices, with particular attention to variables which modified fibrin gel structure and/or its adhesive properties for cells. The variables studied were fibrin concentration, gel cross-linking, and fibronectin and glycosaminoglycan content. Macrophage migration was an inverse linear function of fibrinogen concentration. Little or no fibrinolysis accompanied macrophage migration; rather, macrophages migrated through fibrin gels by an active process associated with marked distortions of cell shape and specialized plasma membrane contacts with fibrin strands. Fibrin matrices prepared from fibrinogen that had been depleted of clotting factor XIII and/or fibronectin provided a superior matrix for macrophage migration. Both the number of migrating cells and distance of migration were reduced when the gel matrix included fibronectin and was cross-linked by factor XIII. A hexapeptide containing the fibronectin cell-binding RGDS sequence reversed this migration inhibition, suggesting that fibronectin immobilized by cross-linking to fibrin may have bound macrophages and restricted cell migration. Hyaluronic acid, heparin, and heparan sulfate inhibited macrophage migration in cross-linked fibrin-fibronectin gels over a range of concentrations. These data are relevant to an understanding of macrophage migration in vivo where cross-linked fibrin-fibronectin gels containing variable amounts of glycosaminoglycans are deposited in tissues in immunologic reactions and in many other types of pathology.     

Involvement of matrix metalloproteinases 2 and 9, tissue inhibitor of metalloproteinases and apoptosis in tissue remodelling in the sheep placenta

Placental growth and development is crucial for successful pregnancy. The aim of this study was to characterize the activity and localization of the matrix metalloproteinase 2 (MMP-2) and MMP-9, which are capable of degrading basement membrane collagen (predominantly collagen type IV), and their endogenous tissue inhibitor of matrix metalloproteinases (TIMPs), in amniotic fluid and in the developing ovine placenta. Cell deletion by apoptosis during placental development was also examined. Zymography with gelatin as substrate indicated that MMP-2 (72 kDa gelatinase A; predominantly latent form) was present in increasing amounts in amniotic fluid from day 70 of gestation to labour (days 140-145), and MMP-9 (92 kDa gelatinase B; predominantly latent form) was detectable from day 125 to labour; there was no increase in MMP-2 or -9 in labour. A broad range of TIMPs was detected in amniotic fluid; the molecular masses corresponded to TIMP-1, -2 and -3. Immunohistochemical techniques localized MMP-2, MMP-9 and TIMP-3 in the sheep placenta, predominantly in the trophoblast layer in uninucleate, but not binucleate, cells. However, MMP-2 and -9 activated proteins in placental homogenates were low throughout pregnancy. Apoptosis was identified by morphological criteria and also by TdT-mediated dUTP nick end labelling. Apoptosis was present in discrete regions in the placenta, predominantly in trophoblast cells near the tips and the basal regions of the fetomaternal interdigitations. During pregnancy the sheep placenta becomes more complex and the area of the fetomaternal interface increases. MMP-2 and -9 are likely to be involved in breaking down basement membranes to allow cell migration during this process. It is suggested that digestion of supporting extracellular matrix may trigger apoptosis and in some way increase the branching pattern in the villi.     

Migration pattern, morphology and viability of cells suspended in or sealed with fibrin glue: a histomorphologic study

INTRODUCTION: We studied the migration pattern, morphology and viability of cells suspended in five different fibrin glues. Besides this, the behaviour of chondrocytes seeded on porous matrices comprising different collagen types sealed with fibrin glue was investigated. MATERIAL AND METHODS: In an experiment A, cell suspension (0.5x10(6) cells) was incubated with different fibrin glues. Experiment B was set up to evaluate chondrocytes migration either through a collagen I/III (Chondro-Gide, Geistlich Biomaterials, Switzerland) or collagen II matrix sealed with different fibrin glues in a perfusion chamber system. Analysis were performed by lightmicroscopy (Mayer's hematoxylin-eosin; Masson-Goldner; TUNEL test) and by transmission and scanning electron microscopy. All fibrin glues were measured for TGF-beta 1 and 2 with a specific ELISA. RESULTS: After incubation of cell suspension in autologous fibrin glue, the morphology of cells is chondrocyte-like. Spindly, process-bearing cells were seen in commercial fibrin glue. Cells suspended in commercial fibrin glue revealed a significant higher percentage of TUNEL positive cells compared to fibrin tissue adhesives mixed with autologous serum (p=0.006). The TGF-beta 1 and 2 concentration was significantly higher in partial autologous fibrin sealant (PAF) compared to their commercial counterparts (p=0.001). Cells seeded on the collagen I/III matrix retained their chondrocytic morphology, while in the type II collagen matrix the chondrocytes displayed a fibroblastic phenotype. The ratio of TUNEL positive cells for the collagen I/III matrix was significantly surpassed by the values, when a collagen II matrix was used (p=0.008). No ingrowth of cells was seen in any of the experimental conditions. CONCLUSION: Partial autologous fibrin glue and collagen I/III matrices are favourable in respect to migration pattern, morphology and viability, but definitive conclusions can only be drawn after in vivo studies. This will be addressed in future animal studies.     

Enzymatic processing of collagen IV by MMP-2 (gelatinase A) affects neutrophil migration and it is modulated by extracatalytic domains

Proteolytic degradation of basement membrane influences the cell behavior during important processes, such as inflammations, tumorigenesis, angiogenesis, and allergic diseases. In this study, we have investigated the action of gelatinase A (MMP-2) on collagen IV, the major constituent of the basement membrane. We have compared quantitatively its action on the soluble forms of collagen IV extracted with or without pepsin (from human placenta and from Engelbreth-Holm-Swarm [EHS] murine sarcoma, respectively). The catalytic efficiency of MMP-2 is dramatically reduced in the case of the EHS murine sarcoma with respect to the human placenta, probably due to the much tighter packing of the network which renders very slow the speed of the rate-limiting step. We have also enquired on the role of MMP-2 domains in processing collagen IV. Addition of the isolated collagen binding domain, corresponding to the fibronectin-like domain of whole MMP-2, greatly in hibits the cleavage process, demonstrating that MMP-2 interacts with collagen type IV preferentially through its fibronectin-like domain. Conversely, the removal of the hemopexin-like domain, using only the catalytic domain of MMP-2, has only a limited effect on the catalytic efficiency toward collagen IV, indicating that the missing domain does not have great relevance for the overall mechanism. Finally, we have investigated the effect of MMP-2 proteolytic activity ex vivo. MMP-2 action negatively affects the neutrophils' migration across type IV coated membranes and this is likely related to the production of lower molecular weight fragments that impair the cellular migration.