STAT3 signalling pathway is implicated in keloid pathogenesis by preliminary transcriptome and open chromatin analyses.

Keloids are wounding-induced fibroproliferative human tumor-like skin scars of complex genetic makeup and poorly defined pathogenesis. To reveal dynamic epigenetic and transcriptome changes of keloid fibroblasts, we performed RNA-seq and ATAC-seq analysis on an early passage keloid fibroblast cell strain and its paired normal control fibroblasts. This keloid strain produced keloid-like scars in a plasma clot-based skin equivalent humanized keloid animal model. RNA-seq analysis reveals gene ontology terms including hepatic fibrosis, Wnt-ß-catenin, TGF-ß, regulation of epithelial-mesenchymal transition (EMT), STAT3 and adherens junction. ATAC-seq analysis suggests STAT3 signalling is the most significantly enriched gene ontology term in keloid fibroblasts, followed by Wnt signalling (Wnt5) and regulation of the EMT pathway. Immunohistochemistry confirms that STAT3 (Tyr705 phospho-STAT3) is activated and ß-catenin is up-regulated in the dermis of keloid clinical specimens and keloid skin equivalent implants from the humanized mouse model. A non-linear dose-response of cucurbitacin I, a selective JAK2/STAT3 inhibitor, in collagen type I expression of keloid-derived plasma clot-based skin equivalents implicates a likely role of STAT3 signalling in keloid pathogenesis. This work also demonstrates the utility of the recently established humanized keloid mouse model in exploring the mechanism of keloid formation.

Keloid-derived, plasma/fibrin-based skin equivalents generate de novo dermal and epidermal pathology of keloid fibrosis in a mouse model.

Keloids are wounding-induced tumor-like human scars. Unclear etiology and lack of animal models to reveal disease mechanisms and invent therapies deepen the grievous health and psychosocial state of vulnerable individuals. Epitomizing the injury-repair environment which triggers and fosters keloid formation and essential dermal/epidermal interactions in disease development, the novel animal model was established by implanting porous polyethylene ring-supported plasma/fibrin-based epidermal-dermal skin constructs on the dorsum of athymic NU/J mice. The implants were stable to 18 weeks, contained abundant human cells, and remodeled to yield scar architecture characteristic of keloid fibrosis compared with normal implants and clinical specimens: (1) macroscopic convex or nodular scar morphology; (2) morphogenesis and accumulation of large collagen bundles from collagen-null initial constructs; (3) epidermal hyperplasia, aberrant epidermal-dermal patency, and features of EMT; (4) increased vasculature, macrophage influx, and aggregation; and (5) temporal-spatial increased collagen-inducing PAI-1 and its interactive partner uPAR expression. Development of such pathology in the NU/J host suggests that T-cell participation is less important at this stage than at keloid initiation. These accessible implants also healed secondary excisional wounds, enabling clinically relevant contemporaneous wounding and treatment strategies, and evaluation. The model provides a robust platform for studying keloid formation and testing knowledge-based therapies.

Leptin of dermal adipose tissue is differentially expressed during the hair cycle and contributes to adipocyte-mediated growth inhibition of anagen-phase vibrissa hair.

Adipose tissue encircles the lower portion of anagen hair follicles and may regulate hair cycle progression. As leptin is a major adipokine, its level of expression from the dermal white adipose tissue during hair cycle progression was studied. The result shows that leptin level is differentially expressed during hair cycle, the lowest in early anagen phase, upregulated in late anagen phase and the highest in the telogen phase. On the other hand, leptin receptor is detected in keratin 15-positive hair bulge epithelium of both anagen- and telogen-phase hair follicles of mice pelage and vibrissa hair, and hair from human scalp. Leptin contributes to adipocyte-mediated growth inhibition of anagen-phase vibrissa hair as demonstrated in organ culture and coculture system. Our data suggest that leptin of dermal white adipose tissue might regulate hair growth and, therefore, hair cycle progression via leptin receptor on the hair follicle epithelium.

PDGF signaling is required for epicardial function and blood vessel formation in regenerating zebrafish hearts.

A zebrafish heart can fully regenerate after amputation of up to 20% of its ventricle. During this process, newly formed coronary blood vessels revascularize the regenerating tissue. The formation of coronary blood vessels during zebrafish heart regeneration likely recapitulates embryonic coronary vessel development, which involves the activation and proliferation of the epicardium, followed by an epithelial-to-mesenchymal transition. The molecular and cellular mechanisms underlying these processes are not well understood. We examined the role of PDGF signaling in explant-derived primary cultured epicardial cells in vitro and in regenerating zebrafish hearts in vivo. We observed that mural and mesenchymal cell markers, including pdgfrß, are up-regulated in the regenerating hearts. Using a primary culture of epicardial cells derived from heart explants, we found that PDGF signaling is essential for epicardial cell proliferation. PDGF also induces stress fibers and loss of cell-cell contacts of epicardial cells in explant culture. This effect is mediated by Rho-associated protein kinase. Inhibition of PDGF signaling in vivo impairs epicardial cell proliferation, expression of mesenchymal and mural cell markers, and coronary blood vessel formation. Our data suggest that PDGF signaling plays important roles in epicardial function and coronary vessel formation during heart regeneration in zebrafish.

Wound healing in development.

Wound healing is the inherent ability of an organism to protect itself against injuries. Cumulative evidence indicates that the healing process patterns in part embryonic morphogenesis and may result in either organ regeneration or scarring, phenomena that are developmental stage- or age-dependent. Skin is the largest organ. Its morphogenesis and repair mechanisms have been studied extensively due not only to its anatomical location, which allows easy access and observation, but also to its captivating structure and vital function. Thus, this review will focus on using skin as a model organ to illustrate new insights into the mechanisms of wound healing that are developmentally regulated in mammals, with special emphasis on the role of the Wnt signaling pathway and its crosstalk with TGF-ß signaling. Relevant information from studies of other organs is discussed where it applies, and the clinical impact from such knowledge and emerging concepts on regenerative medicine are discussed in perspective.

Heart repair and regeneration: recent insights from zebrafish studies.

Cardiovascular disease is the leading cause of death in the U.S. and worldwide. Failure to properly repair or regenerate damaged cardiac tissues after myocardial infarction is a major cause of heart failure. In contrast to humans and other mammals, zebrafish hearts regenerate after substantial injury or tissue damage. Here, we review recent progress in studying zebrafish heart regeneration, addressing the molecular and cellular responses in the three tissue layers of the heart: myocardium, epicardium, and endocardium. We also compare different injury models utilized to study zebrafish heart regeneration and discuss the differences in responses to injury between mammalian and zebrafish hearts. By learning how zebrafish hearts regenerate naturally, we can better design therapeutic strategies for repairing human hearts after myocardial infarction.

From buds to follicles: matrix metalloproteinases in developmental tissue remodeling during feather morphogenesis.

Organogenesis involves a series of dynamic morphogenesis and remodeling processes. Since feathers exhibit complex forms, we have been using the feather as a model to analyze how molecular pathways and cellular events are used. While several major molecular pathways have been studied, the roles of matrix degrading proteases and inhibitors in feather morphogenesis are unknown. Here we addressed this knowledge gap by studying the temporal and spatial expression of proteases and inhibitors in developing feathers using mammalian antibodies that cross react with chicken proteins. We also investigated the effect of protease inhibitors on feather development employing an in vitro feather bud culture system. The results show that antibodies specific for mammalian MMP2 and TIMP2 stained positive in both feather epithelium and mesenchyme. The staining co-localized in structures of E10-E13 developing feathers. Interestingly, MMP2 and TIMP2 exhibited a complementary staining pattern in developing E15 and E20 feathers and in maturing feather filaments. Although they exhibited a slight delay in feather bud development, similar patterns of MMP2 and TIMP2 staining were observed in in vitro culture explants. The broad spectrum pharmacological inhibitors AG3340 and BB103 (MMP inhibitors) but not Aprotinin (a plasmin inhibitor) showed a reversible effect on epithelium invagination and feather bud elongation. TIMP2, a physiological inhibitor to MMPs, exhibited a similar effect. Markers of feather morphogenesis showed that MMP activity was required for both epithelium invagination and mesenchymal cell proliferation. Inhibition of MMP activity led to an overall delay in the expression of molecules that regulate either early feather bud growth and/or differentiation and thereby produced abnormal buds with incomplete follicle formation. This work demonstrates that MMPs and their inhibitors are not only important in injury repair, but also in development tissue remodeling as demonstrated here for the formation of feather follicles.

Adenoviral overexpression and small interfering RNA suppression demonstrate that plasminogen activator inhibitor-1 produces elevated collagen accumulation in normal and keloid fibroblasts.

Keloids are tumor-like skin scars that grow as a result of the aberrant healing of skin injuries, with no effective treatment. We provide new evidence that both overexpression of plasminogen activator inhibitor-1 (PAI-1) and elevated collagen accumulation are intrinsic features of keloid fibroblasts and that these characteristics are causally linked. Using seven strains each of early passage normal and keloid fibroblasts, the keloid strains exhibited inherently elevated collagen accumulation and PAI-1 expression in serum-free, 0.1% ITS+ culture; larger increases in these parameters occurred when cells were cultured in 3% serum. To demonstrate a causal relationship between PAI-1 overexpression and collagen accumulation, normal fibroblasts were infected with PAI-1-expressing adenovirus. Such cells exhibited a two- to fourfold increase in the accumulation of newly synthesized collagen in a viral dose-dependent fashion in both monolayers and fibrin gel, provisional matrix-like cultures. Three different PAI-1-targeted small interfering RNAs, alone or in combination, produced greater than an 80% PAI-1 knockdown and reduced collagen accumulation in PAI-1-overexpressing normal or keloid fibroblasts. A vitronectin-binding mutant of PAI-1 was equipotent with wild-type PAI-1 in inducing collagen accumulation, whereas a complete protease inhibitor mutant retained approximately 50% activity. Thus, PAI-1 may use more than its protease inhibitory activity to control keloid collagen accumulation. PAI-1-targeted interventions, such as small interfering RNA and lentiviral short hairpin RNA-containing microRNA sequence suppression reported here, may have therapeutic utility in the prevention of keloid scarring.

Accelerated closure of skin wounds in mice deficient in the homeobox gene Msx2.

Differences in cellular competence offer an explanation for the differences in the healing capacity of tissues of various ages and conditions. The homeobox family of genes plays key roles in governing cellular competence. Of these, we hypothesize that Msx2 is a strong candidate regulator of competence in skin wound healing because it is expressed in the skin during fetal development in the stage of scarless healing, affects postnatal digit regeneration, and is reexpressed transiently during postnatal skin wound repair. To address whether Msx2 affects cellular competence in injury repair, 3 mm full-thickness excisional wounds were created on the back of C.Cg-Msx2(tm1Rilm)/Mmcd (Msx2 null) mice and the healing pattern was compared with that of the wild type mice. The results show that Msx2 null mice exhibited faster wound closure with accelerated reepithelialization plus earlier appearance of keratin markers for differentiation and an increased level of smooth muscle actin and tenascin in the granulation tissue. In vitro, keratinocytes of Msx2 null mice exhibit increased cell migration and the fibroblasts show stronger collagen gel contraction. Thus, our results suggest that Msx2 regulates the cellular competence of keratinocytes and fibroblasts in skin injury repair.

Regeneration of fat cells from myofibroblasts during wound healing.

Although regeneration through the reprogramming of one cell lineage to another occurs in fish and amphibians, it has not been observed in mammals. We discovered in the mouse that during wound healing, adipocytes regenerate from myofibroblasts, a cell type thought to be differentiated and nonadipogenic. Myofibroblast reprogramming required neogenic hair follicles, which triggered bone morphogenetic protein (BMP) signaling and then activation of adipocyte transcription factors expressed during development. Overexpression of the BMP antagonist Noggin in hair follicles or deletion of the BMP receptor in myofibroblasts prevented adipocyte formation. Adipocytes formed from human keloid fibroblasts either when treated with BMP or when placed with human hair follicles in vitro. Thus, we identify the myofibroblast as a plastic cell type that may be manipulated to treat scars in humans.

Novel skin chamber for rat ischemic flap studies in regenerative wound repair.

BACKGROUND: In plastic surgery, skin flap is an important approach to reconstructive wound repairs. The rat dorsal skin flap is a clinically relevant and popular animal model to investigate and evaluate flap survival and necrosis. Nonetheless, flap survival is often unstable with unpredictable outcomes, regardless of previous attempts at design modification. METHODS & RESULTS: In the present study, we report a novel flap chamber that provides stable and reproducible outcomes by separating the dorsal skin flap from its surrounding skin by in situ immobilization. The flap chamber blocks circulation that disturbs flap ischemia from both basal and lateral sides of the flap tissue. Demarcation of skin necrosis is macroscopically evident on the flap and supported by distinct changes in histological architecture under microscopic examination. The utility of the novel skin flap chamber is further proven by applying it to the examination of flap survival in streptozotocin-induced diabetic rats with an increase in skin necrosis. The flap chamber also affords size modifications where a narrower flap chamber increases ischemia and provides manipulable therapeutic windows for studying cell therapies. Accordingly, intradermal injection of endothelial cells 3 days before flap ischemia significantly increases the survival of skin flaps. CONCLUSIONS: The novel flap chamber not only may stabilize the skin flap and provide reproducible outcomes that overcome the shortfalls of the traditional ischemic flap but also may afford size modifications that support research designs and test therapeutic approaches to regenerative repair.

Mechanisms of hypoxic regulation of plasminogen activator inhibitor-1 gene expression in keloid fibroblasts.

Keloids are an excessive accumulation of extracellular matrix. Although numerous studies have shown elevated plasminogen activator inhibitor-1 (PAI-1) levels in keloid fibroblasts compared with those of normal skin. Their specific mechanisms involved in the differential expression of PAI-1 in these cell types. In this study, the upregulation of PAI-1 expression is demonstrated in keloid tissues and their derived dermal fibroblasts, attesting to the persistence, if any, of fundamental differences between in vivo and in vitro paradigms. We further examined the mechanisms involved in hypoxia-induced regulation of PAI-1 gene in dermal fibroblast derived from keloid lesions and associated clinically normal peripheral skins from the same patient. Primary cultures were exposed to an environmental hypoxia or desferroxamine. We found that the hypoxia-induced elevation of PAI-1 gene appears to be regulated at both transcriptional and post-transcriptional levels in keloid fibroblasts. Furthermore, our results showed a consistent elevation of HIF-1alpha protein level in keloid tissues compared with their normal peripheral skin controls, implying a potential role as a biomarker for local skin hypoxia. Treatment with antisense oligonucleotides against hypoxia-inducible factor 1alpha (HIF-1alpha) led to the downregulation of steady-state levels of PAI-1 mRNA under both normoxic and hypoxic conditions. Conceivably, our results suggest that HIF-1alpha may be a novel therapeutic target to modulate the scar fibrosis process.

Evidence for recipient derived fibroblast recruitment and activation during the development of chronic cardiac allograft rejection.

BACKGROUND: Allograft fibrosis is a prominent feature of chronic rejection. Although intragraft fibroblasts contribute to this process, their origin and exact role remain poorly understood. METHODS: Using a rat model of chronic rejection, LEW to F344, cardiac fibroblasts were isolated at the point of rejection and examined in a collagen gel contraction assay to measure fibroblast activation. The allograft microenvironment was examined using immunohistochemistry for fibrogenic markers (transforming growth factor [TGF]-beta, platelet-derived growth factor [PDGF], tissue plasminogen activator [TPA], plasminogen activator inhibitor [PAI]-1, matrix metalloproteinase [MMP]-2, and tissue inhibitor of matrix metalloproteinase [TIMP]-2). The origin of intragraft fibroblasts was studied using female to male allografts followed by polymerase chain reaction [PCR] and in situ hybridization for the male sry gene. RESULTS: The cardiac fibroblasts isolated from allografts with chronic rejection exhibited higher gel contractibility (50.9% +/- 6.1% and 68.2% +/- 3.8% at 4 and 24 hr) compared with naive cardiac fibroblasts (30.7% +/- 3.5% and 55.3% +/- 6.6% at 4 and 24 hr; P<0.05 and <0.05, respectively). Immunostaining for TGF-beta, PDGF, TPA, PAI-1, MMP-2 and TIMP-2 was observed in all allografts at the time of rejection. In situ hybridization demonstrated the presence of sry positive cells in female allografts rejected by male recipients. Sixty-five percent of fibroblast colonies (55 of 85) isolated from female heart allografts expressed the male sry gene. CONCLUSION: Cardiac fibroblasts are activated and exist in a profibrogenic microenvironment in allografts undergoing chronic rejection. A substantial proportion of intragraft fibroblasts are recruited from allograft recipients in this experimental model of chronic cardiac allograft rejection.

Activation of NFkappaB signal pathways in keloid fibroblasts.

Keloids are characterized as an "over-exuberant" healing response resulting in a disproportionate extracellular matrix (ECM) accumulation and tissue fibrosis. In view of the integral role of inflammation and cytokines in the healing response, it is logical to assume that they may play a part in orchestrating the pathology of this "abnormal" healing process. Tumor necrosis factor-alpha (TNF-alpha) is a potent proinflammatory cytokine involved in activation of signaling events and transcriptional programs, such as NFkappaB. This study attempts to determine the difference in NFkappaB and its related genes expression and DNA binding activity between keloid and normal skin fibroblasts. Three keloid and normal skin tissues (NSk) and their derived fibroblasts were used to determine NFkappaB signaling pathway expression using specific cDNA microarrays, Western blot analysis and immunohistochemistry. Electrophoretic mobility gel shift assay (EMSA) was used to assess NFkappaB-binding activity, all assays were performed in the presence and absence of TNF-alpha. TNF-alpha up-regulated 15% of NFkappaB signal pathway related genes in keloid fibroblast compared to normal skin. At the protein level, keloid fibroblasts and tissues showed higher basal levels of TNF- receptor-associated factors-TRAF1, TRAF2-TNF-alpha, inhibitor of apoptosis (c-IAP-1), and NFkappaB, compared with NSk. Keloid fibroblasts showed a constitutive increase in NFkappaB-binding activity in comparison to NSk both with and without TNF-alpha treatment. NFkappaB and its targeted genes, especially the antiapoptotic genes, could play a role in keloid pathogenesis; targeting NFkappaB could help in developing therapeutic interventions for the treatment of keloid scarring.

Extracellular matrix domain formation as an indicator of chondrocyte dedifferentiation and hypertrophy.

Cartilage injury represents one of the most significant clinical conditions. Implantation of expanded autologous chondrocytes from noninjured compartments of the joint is a typical strategy for repairing cartilage. However, two-dimensional culture causes dedifferentiation of chondrocytes, making them functionally inferior for cartilage repair. We hypothesized that functional exclusion of dedifferentiated chondrocytes can be achieved by the selective mapping of collagen molecules deposited by chondrogenic cells in a three-dimensional environment. Freshly isolated and in vitro expanded human fetal or adult articular chondrocytes were cultured in a thermoreversible hydrogel at density of 1 × 10(7) cells/mL for 24 h. Chondrocytes were released from the gel, stained with antibodies against collagen type 2 (COL II) or COL I or COL X and sorted by fluorescence activated cell sorting. Imaging flow cytometry, immunohistochemistry, quantitative polymerase chain reaction, and glycosaminoglycan (GAG) assays were performed to evaluate the differences between COL II domain forming and COL II domain-negative cells. Freshly dissected periarticular chondrocytes robustly formed domains that consisted of the extracellular matrix surrounding cells in the hydrogel as a capsule clearly detectable by imaging flow cytometry (ImageStream) and confocal microscopy. These domains were almost exclusively formed by COL II. In contrast to that, a significant percentage of freshly isolated growth plate pre-hypertrophic and hyperdrophic chondrocytes deposited matrix domains positive for COL II, COL I, and COL X. The proportion of the cells producing COL II domains decreased with the increased passage of in vitro expanded periarticular fetal or adult articular chondrocytes. Sorted COL II domain forming cells deposited much higher levels of COL II and GAGs in pellet assays than COL II domain-negative cells. COL II domain forming cells expressed chondrogenic genes at higher levels than negative cells. We report a novel method that allows separation of functionally active chondrogenic cells, which deposit high levels of COL II from functionally inferior dedifferentiated cells or hypertrophic chondrocytes producing COL X. This approach may significantly improve current strategies used for cartilage repair.

In vitro culture of epicardial cells from adult zebrafish heart on a fibrin matrix.

We describe here a protocol for culturing epicardial cells from adult zebrafish hearts, which have a unique regenerative capacity after injury. Briefly, zebrafish hearts first undergo ventricular amputation or sham operation. Next, the hearts are excised and explanted onto fibrin gels prepared in advance in a multiwell tissue culture plate. The procedure allows the epicardial cells to outgrow from the ventricle onto a fibrin matrix in vitro. This protocol differs from those used in other organisms by using a fibrin gel to mimic blood clots that normally form after injury and that are essential for proper cell migration. The culture procedure can be accomplished within 5 h; epicardial cells can be obtained within 24-48 h and can be maintained in culture for 5-6 d. This protocol can be used to investigate the mechanisms underlying epicardial cell migration, proliferation and epithelial-to-mesenchymal transition during heart regeneration, homeostatic cardiac growth or other physiological processes.

Memory encoded throughout our bodies: molecular and cellular basis of tissue regeneration.

When a sheep loses its tail, it cannot regenerate it in the manner of lizards. On the other hand, it is possible to clone mammals from somatic cells, showing that a complete developmental program is intact in a wounded sheep's tail the same way it is in a lizard. Thus, there is a requirement for more than only the presence of the entire genetic code in somatic cells for regenerative abilities. Thoughts like this have motivated us to assemble more than just a factographic synopsis on tissue regeneration. As a model, we review skin wound healing in chronological order, and when possible, we use that overview as a framework to point out possible mechanisms of how damaged tissues can restore their original structure. This article postulates the existence of tissue structural memory as a complex distributed homeostatic mechanism. We support such an idea by referring to an extremely fragmented literature base, trying to synthesize a broad picture of important principles of how tissues and organs may store information about their own structure for the purposes of regeneration. Selected developmental, surgical, and tissue engineering aspects are presented and discussed in the light of recent findings in the field. When a sheep loses its tail, it cannot regenerate it in the manner of lizards. On the other hand, it is possible to clone mammals from somatic cells, showing that a complete developmental program is intact in a wounded sheep's tail the same way it is in a lizard. Thus, there is a requirement for more than only the presence of the entire genetic code in somatic cells for regenerative abilities. Thoughts like this have motivated us to assemble more than just a factographic synopsis on tissue regeneration. As a model, we review skin wound healing in chronological order, and when possible, we use that overview as a framework to point out possible mechanisms of how damaged tissues can restore their original structure. This article postulates the existence of tissue structural memory as a complex distributed homeostatic mechanism. We support such an idea by referring to an extremely fragmented literature base, trying to synthesize a broad picture of important principles of how tissues and organs may store information about their own structure for the purposes of regeneration. Selected developmental, surgical, and tissue engineering aspects are presented and discussed in the light of recent findings in the field.

Transforming growth factor-beta3 affects plasminogen activator inhibitor-1 expression in fetal mice and modulates fibroblast-mediated collagen gel contraction.

For over two decades, the precise role of transforming growth factor-beta (TGF-beta) isoforms in scarless healing of mammalian fetal skin wounds has generated much interest. Although their exact role remains to be established, it has been suggested that high TGF-beta3 activity may correlate with a scarless phenotype. Previously, we showed that plasminogen activator inhibitor-1 (PAI-1), a known TGF-beta downstream molecule and marker of fibrosis, is also developmentally regulated during fetal skin development. In this study, the relationship between TGF-beta3 and PAI-1 was investigated using embryonic day 14.5 TGF-beta3 knockout (ko) mice. The results showed increased PAI-1 expression in the epidermis and dermis of ko mice, using an ex vivo limb-wounding study. Furthermore, increased PAI-1 expression and activity was seen in embryo extracts and conditioned media of ko dermal fibroblasts. When TGF-beta3 knockout fibroblasts were placed into three-dimensional collagen matrices, they were found to have decreased collagen gel contraction, suggesting altered cell-matrix interaction. These findings provide a further avenue for the interactive role of TGF-beta3 and PAI-1 during fetal scarless repair.

Plasminogen activator/plasmin system: a major player in wound healing?

The role of the plasminogen activator/plasmin system in fibrinolysis has been well established. Indeed, clinicians worldwide have successfully utilized recombinant tissue-type plasminogen activator as first-line treatment of acute myocardial infarction for almost 2 decades. Outside the field of cardiology, there has been increasing excitement regarding the possible contribution of this system in many other important biological processes, including cell adhesion, cell migration, cell-cell signaling, tumor invasion and metastasis, ovulation, and wound healing. In this review, we present evidence in the current literature that the plasminogen activator/plasmin system does have a role in wound healing, looking at both normal and abnormal healing. Furthermore, the invaluable insights provided by numerous transgenic animal experiments are summarized.

Fibrinogen inhibits fibroblast-mediated contraction of collagen.

Extracellular matrix changes in composition and organization as it transitions from the provisional matrix of the fibrin/platelet plug to collagen scar in healed wounds. The manner in which individual matrix proteins affect these activities is not well established. In this article we describe the interactions of two important extracellular matrix components, fibrin and collagen, using an in vitro model of wound contraction, the fibroblast-populated collagen lattice. We utilized different fibrinogen sources and measured tissue reorganization in floating and tensioned collagen lattices. Our results showed that both fibrin and fibrinogen decreased the contraction of fibroblast populated collagen lattices in a dose-dependent manner. Polymerization of fibrinogen to fibrin using thrombin had no effect on this inhibition. Further, there was no effect due to changes in protein concentration, alternate components of the fibrin sealant, or the enzymatic action of thrombin. These results suggest that the initial stability of the fibrin provisional matrix is due to the fibrin, because this protein appears to inhibit contraction of the matrix. This may be important in the early phases of wound healing when clot stability is vital for hemostasis. Later, as fibrin is replaced by collagen, wound contraction can occur.