Provisional Fibrin Matrix Research Articles

A novel in vitro dermal wound healing model incorporating a response to mechanical wounding and repopulation of a fibrin provisional matrix

A novel in vitro dermal wound healing model incorporating a response to mechanical wounding and repopulation of a fibrin provisional matrix

IL-8-Stimulated Expression of Urokinase-Type Plasminogen Activator in Human Skin and Human Epidermal Cells

Extracellular matrix (ECM) remodeling is essential for normal development and tissue repair. Although many roles for extracellular proteinases in the breakdown of ECM have been established, the regulations of these proteinases in human tissue are not fully understood. Inflammatory cytokines have been implicated in the regulation of several matrix metalloproteinases. To determine whether these mediators have a similar effect on fibrinolysis and the remodeling of the fibrin provisional matrix, we examined the role of cytokines on the regulation of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) in human skin. In this report, we show that interleukin-8 (IL-8), but not other cytokines tested, is a potent inducer of the 38-kDa uPA in organ-cultured human skin. In addition, the uPA inhibitor, PAI-1, was not affected by IL-8. When primary epidermal human keratinocytes were treated with IL-8, 55-kDa pro-uPA was significantly induced in the conditioned medium. The mRNA expression of uPA in the keratinocytes was found to be constitutively elevated and was not affected by IL-8. To support such a notion, activation of the 5′-flanking promoter of the human uPA gene was measured using the CAT reporter assay. Consistent with the results of mRNA measurement, the promoter is constitutively active in keratinocytes and is not affected by IL-8. In contrast, the promoter construct is neither active in the dermal fibroblasts nor stimulated by the cytokine. This differential transactivation of uPA gene in these cells indicates that keratinocyte-specific factors may govern the basal expression of the gene. These results indicate a complex regulation of uPA expression in epidermal cells.

PAI-1, fibrosis, and the elusive provisional fibrin matrix

Whether induced surgically or by hypertension, infections, extreme heat, or caustic chemicals, tissue injury invariably leads to vasodilatation, with subsequent leakage of plasma proteins into the connective tissues, rapid activation of the coagulation cascade, and deposition of fibrin. A central paradigm in the field is that the fibrin is organized into a “provisional fibrin matrix,” which acts as a road map to direct the migration of invading cells. Leukocytes and possibly fibroblasts migrate into the area and elaborate cytokines which, in turn, stimulate resident cells to synthesize and deposit collagens and other insoluble fibrillar components into the evolving extracellular matrix (ECM). Fibrotic disease occurs when normal control of this process is compromised and excess fibrous material accumulates in the tissues. It is generally assumed that the persistence of fibrin in the matrix promotes fibrosis, and that the extent of fibrosis is limited by proteinases that remove the fibrin (i.e., the fibrinolytic system). In a recent issue of the JCI, Hattori et al. (1) affirm previous suggestions that plasminogen activator inhibitor-1 (PAI-1) promotes pathological fibrosis but challenges the concept that fibrin is required.

Role of α5β1 and αvβ3 Integrins on Smooth Muscle Cell Spreading and Migration in Fibrin Gels

Fibrin is found at sites of vascular injury and is one of the major matrix ligands for beta3 integrins. Blocking the beta3 integrin on smooth muscle cell is hypothesized as a potential target to prevent restenosis because it could inhibit cell attachment and migration into fibrin provisional matrix. Human aortic smooth muscle cells (HNB18E6E7) spread stably in plasma gels within 24 h. Cell spreading was dramatically blocked by simultaneous use of alpha5beta1 and alphavbeta3 integrin antibodies (P <0.0001), however, blocking of either integrin alone failed to inhibit spreading. GPenGRGDSPCA, which has been considered a specific alphavbeta3 antagonist, inhibited spreading at 500 microM, suggesting that the peptide blocked both alpha5beta1 and alphavbeta3. Similarly, invasive migration into fibrin gels was blocked by simultaneous use of both alpha5beta1 and alphavbeta3 antibodies, however, blocking of either integrin alone failed to effect cell migration. Another migration assay using transwell indicated similar results. In conclusion, both alpha5beta1 and alphavbeta3 integrins are responsible for smooth muscle cell spreading and migration into fibrin gels. These data suggest that blocking beta3 integrin alone would not affect smooth muscle cell interaction with fibrin.

Tissue transglutaminase is expressed, active, and directly involved in rat dermal wound healing and angiogenesis.

Tissue transglutaminase (TG) is an enzyme that stabilizes the structure of tissues by covalently ligating extracellular matrix molecules. Expression and localization of TG are not well established during wound healing. We performed punch biopsy wounds on anesthetized rats and monitored the wound healing process by histological and immunohistochemical methods. The TG antigen and activity are expressed at sites of neovascularization in the provisional fibrin matrix within 24 h of wounding. Endothelial cells, macrophages, and skeletal muscle cells expressed TG throughout the healing process. The TG antigen within the wound was active in vivo based on the detection of isopeptide bonds. The TG antigen increased four- to fivefold by day 3 postwounding and was proteolytically degraded. TG expression occurred in association with TGF-beta, TNF-alpha, IL-6, and VEGF production in the wound. Recombinant TG increased vessel length density (a measure of angiogenesis) when applied topically in rat dorsal skin flap window chambers. We have established that TG is an important tissue stabilizing enzyme that is active during wound healing and can function to promote angiogenesis.

Expression of Wnt genes in early wound healing.

The Wnt family of developmental genes has previously been shown to be involved in proliferation, differentiation, and cell to cell signaling during embryogenesis. In addition, several Wnt genes have been shown to be expressed during carcinogenesis. We have investigated these genes during the wound-healing process. Wnt-4 gene expression is found in mouse wounds from 2 hours to 30 hours postwounding. The expression of Wnt-4 is also stimulated by direct trauma to murine fibroblasts in culture, and the expression is greatly enhanced by the addition of a short plasmin digest of fibrin. Therefore the regulation of Wnt-4, appears to be complex, with expression being stimulated both by direct trauma and by the influence of clotting and fibrinolysis products. We propose that the expression of Wnt-4 in the early wound, in response to the provisional fibrin matrix, regulates cell movement and proliferation in the creation of new tissue by mechanisms related to those of embryogenesis.

Acute lung injury fibroblast migration and invasion of a fibrin matrix is mediated by CD44.

Fibrosis results when myofibroblasts invade the wound fibrin provisional matrix. Extracellular matrix receptors on the cell surface mediate cell adhesion, migration, and invasion. Recent work with transformed cells indicates that these cells use the cell surface matrix receptor CD44 for migration and invasion. In this study, we examine whether lung fibroblasts, isolated from patients dying with acute alveolar fibrosis, use CD44 to invade a fibrin matrix. Consistent with a role for CD44 in mediating fibroblast invasion and subsequent tissue fibrosis, immunohistochemical analysis of lung tissue from patients who died from acute alveolar fibrosis after lung injury reveals CD44-expressing mesenchymal cells throughout newly formed fibrotic tissue. PCR, Western, and immunoprecipitation analysis demonstrate that the 85-kD CD44 isoform is expressed by acute lung injury fibroblasts. Consistent with a role in mediating matrix adhesion and migration ultrastructurally, CD44 was found uniformly over the cell surface and was found densely labeling filopodia and lamellipodia, highly motile structures involved in cell migration. To determine if lung injury fibroblasts use CD44 to invade fibrin, a fibrin gel model of fibrosis was used. By blocking the function of CD44 with monoclonal antibodies, fibroblast invasion into a fibrin matrix was inhibited. To examine the mechanism by which CD44 mediates fibroblast invasion, the role of CD44 in fibroblast migration and adhesion was evaluated. Anti-CD44 antibody blocked fibroblast migration on the provisional matrix proteins fibronectin, fibrinogen, and hyaluronic acid. Additionally, fibroblast CD44 mediated adhesion to the provisional matrix proteins fibronectin, fibrin, and hyaluronic acid, but not to laminin, a component of the basement membrane. These findings support the hypothesis that fibroblast CD44 functions as an adhesion receptor for provisional matrix proteins and is capable of mediating fibroblast migration and invasion of the wound provisional matrix resulting in the formation of fibrotic tissue.

CD44-related chondroitin sulfate proteoglycan, a cell surface receptor implicated with tumor cell invasion, mediates endothelial cell migration on fibrinogen and invasion into a fibrin matrix.

Microvascular endothelial cell invasion into the fibrin provisional matrix is an integral component of angiogenesis during wound repair. Cell surface receptors which interact with extracellular matrix proteins participate in cell migration and invasion. Malignant cells use CD44-related chondroitin sulfate proteoglycan (CSPG) as a matrix receptor to mediate migration and invasion. In this study, we examine whether cell surface CSPG can mediate similar events in nonmalignant wound microvascular endothelial cells or whether use of CSPG for migration and invasion is a property largely restricted to malignant cells. After inhibiting CSPG synthesis with p-nitrophenyl beta-d xylopyranoside (beta-d xyloside), wound microvascular endothelial cells were capable of attaching and spreading on the surface of a fibrin gel; however, their ability to invade the fibrin matrix was virtually eliminated. To begin to examine the mechanism by which endothelial cells use CSPG to invade fibrin matrices, cell adhesion and migration on fibrinogen was examined. Endothelial cell adhesion and migration on fibrinogen were inhibited by both beta-d xyloside and after cleavage of chondroitin sulfate from the core protein by chondroitinase ABC. We have determined that wound microvascular endothelial cells express the majority of their proteoglycan as CSPG and that the CSPG core protein is immunologically related to CD44. PCR studies show that these cells express both the "standard" (CD44H) isoform and an isoform containing the variably spliced exon V3. In addition, anti-CD44 antibody blocks endothelial cell migration on fibrinogen. Affinity chromatography studies reveal that partially purified microvascular endothelial cell CSPG binds fibrinogen. These findings suggest that CD44-related CSPG, a molecule implicated in the invasive behavior of tumor cells, is capable of binding fibrinogen/fibrin, thereby mediating endothelial cell migration and invasion into the fibrin provisional matrix during wound repair.

Alpha 2-Antiplasmin and plasminogen activator inhibitors in healing human skin wounds.

Mechanical injury of tissues is followed by the formation of a provisional fibrin matrix, which is later replaced by granulation tissue. The fibrinolytic proteinase, plasmin, is thought to contribute to the displacement of the primary matrix. Plasmin is generated from the ubiquitous proenzyme plasminogen by plasminogen activators. The system of plasminogen activation is controlled at several levels: plasminogen activator inhibitors (PAI-1 and PAI-2) counteract the activity of plasminogen activators and alpha 2-antiplasmin inhibits the activity of plasmin. In order to elucidate the mechanisms that regulate the plasminogen activator system in healing human skin wounds, we performed the immunohistological study reported here. The plasmin inhibitor alpha 2-antiplasmin and PAI-2 were found in the primary fibrin-rich matrix and in the granulation tissue. alpha 2-Antiplasmin was diffusely distributed in the tissue and its distribution correlated with the presence and localization of plasmin(ogen) except that, in contrast to plasmin(ogen), the alpha 2-antiplasmin was apparently not cell-associated. The stainings for PAI-2 increased with time and paralleled the development of the cellular infiltrate. PAI-2 was found in association with cells, which were identified by double immunofluorescence stainings as monocytes/macrophages and fibroblasts. In line with the immunohistological data, polymerase chain reaction after reverse transcription revealed mRNA for PAI-2 in healing human skin wounds. Taken together, our findings indicate that in healing human skin wounds, PAI-2 is the primary regulator of plasminogen activators, whereas alpha 2-antiplasmin may serve to control plasmin activity.

Accelerated Epithelization Under a Highly Vapor-Permeable Wound Dressing Is Associated with Increased Precipitation of Fibrin(ogen) and Fibronectin

In a previous study we showed that the use of a newly developed, highly water vapor permeable, PEU wound dressing accelerates the epithelization of partial-thickness wounds more than an occlusive wound dressing (OpSite) in comparison with air exposure. The purpose of this study was to investigate the distribution of fibrin(ogen), fibronectin, and type IV collagen during the epithelization process under these three conditions. The breathable PEU film enabled coagulation of the wound exudate, preserving it into a semisolid gelatinous state. This coagulum layer contained an abundant amount of fibrin(ogen) and fibronectin. In wounds occluded with OpSite film, depositions of fibrin(ogen) and fibronectin were less extensive. Migrating keratinocytes contained intracellular depositions of fibrin(ogen), suggesting that these cells phagocytize components of the provisional fibrin matrix during wound healing. It was concluded that accelerated epithelization underneath the highly water vapor permeable polyetherurethane film dressing is associated with the presence of a gelatinous coagulum containing fibrin(ogen) and fibronectin. We speculate that the enhanced healing rate might be caused by an increased concentration of growth-promoting factors present in the residual exudate underneath the PEU dressing.