The role of focal adhesion complexes in fibroblast mechanotransduction during scar formation

Historically, great efforts have been made to elucidate the biochemical pathways that direct the complex process of wound healing; however only recently has there been recognition of the importance that mechanical signals play in the process of tissue repair and scar formation. The body's physiologic response to injury involves a dynamic interplay between mechanical forces and biochemical cues which directs a cascade of signals leading ultimately to the formation of fibrotic scar. Fibroblasts are a highly mechanosensitive cell type and are also largely responsible for the generation of the fibrotic matrix during scar formation and are thus a critical player in the process of mechanotransduction during tissue repair. Mechanotransduction is initiated at the interface between the cell membrane and the extracellular matrix where mechanical signals are first translated into a biochemical response. Focal adhesions are dynamic multi-protein complexes through which the extracellular matrix links to the intracellular cytoskeleton. These focal adhesion complexes play an integral role in the propagation of this initial mechanical cue into an extensive network of biochemical signals leading to widespread downstream effects including the influx of inflammatory cells, stimulation of angiogenesis, keratinocyte migration, fibroblast proliferation and collagen synthesis. Increasing evidence has demonstrated the importance of the biomechanical milieu in healing wounds and suggests that an integrated approach to the discovery of targets to decrease scar formation may prove more clinically efficacious than previous purely biochemical strategies.     

The extracellular matrix: an active or passive player in fibrosis?

Fibrosis is characterized by excessive accumulation of collagen and other extracellular matrix (ECM) components, and this process has been likened to aberrant wound healing. The early phases of wound healing involve the formation of a provisional ECM containing fibrin, fibrinogen, and fibronectin. Fibroblasts occupy this matrix and proliferate in response to activators elaborated by leukocytes that have migrated into the wound and are retained by the ECM. This coincides with the appearance of the myofibroblast, a specialized form of fibroblast whose differentiation is primarily driven by cytokines, such as transforming growth factor-β (TGF-β), and by mechanical tension. When these signals are reduced, as when TGF-β secretion is reduced, or as in scar shrinkage, myofibroblasts undergo apoptosis, resulting in a collagen-rich, cell-poor scar. Retention of myofibroblasts in fibrosis has been described as the result of imbalanced cytokine signaling, especially with respect to levels of activated TGF-β. ECM components can regulate myofibroblast persistence directly, since this phenotype is dependent on extracellular hyaluronan, tenascin-C, and the fibronectin splice variant containing the "extra domain A," and also, indirectly, through retention of TGF-β-secreting cells such as eosinophils. Thus the ECM is actively involved in both cellular and extracellular events that lead to fibrosis. Targeting components of the ECM as cells respond to injury and inflammatory stimuli holds promise as a means to avoid development of fibrosis and direct the wound-healing process toward reestablishment of a healthy equilibrium.     

Gene profiling of the rat medial collateral ligament during early healing using microarray analysis

Ligament heals in a synchronized and complex series of events. The remodeling process may last months or years. Experimental evidence suggests the damaged ligament does not recover its normal functional properties. Specific mechanisms to prevent scar formation and to regenerate the original mechanical function remain elusive but likely involve regulation of creeping substitution. Creeping substitution creates a larger hypercellular, hypervascular, and disorganized granulation tissue mass that results in an inefficient and nonregenerative wound healing process for the ligament. Control of creeping substitution may limit the extent of this tissue compromise and reduce the time necessary for healing. The objective of this study is to better understand the mechanism behind scar formation by identifying the extracellular matrix factors and other unique genes of interest differentially expressed during rat ligament healing via microarray. For this study, rat medial collateral ligaments were either surgically transected or left intact. Ligaments were collected at day 3 or 7 postinjury and used for microarray, quantitative PCR, and/or immunohistochemistry. Results were compared with the normal intact ligament. We demonstrate that early ligament healing is characterized by the modulation of several inflammatory and extracellular matrix factors during the first week of injury. Specifically, a number of matrix metalloproteinases and collagens are differentially and significantly expressed during early ligament healing. Additionally, we demonstrate the modulation of three novel genes, periostin, collagen-triple helix repeat containing-1, and serine protease 35 in our ligament healing model. Together, control of granulation tissue creeping substitution and subsequent downstream scar formation is likely to involve these factors.     

Global gene expression profiles reveal an increase in mRNA levels of collagens, MMPs, and TIMPs in late radiation enteritis

Radiation enteritis, a common complication of radiation therapy for abdominal and pelvic cancers, is characterized by severe transmural fibrosis associated with mesenchymal cell activation, tissue disorganization, and deposition of fibrillar collagen. To investigate the mechanisms involved in this pathological accumulation of extracellular matrix, we studied gene expression of matrix components along with that of genes involved in matrix remodeling, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs). Hybrid selection on high-density cDNA array, real-time RT-PCR, gelatin zymography and immunohistochemistry were used to characterize the mRNA expression profile, activity, and tissue location of extracellular matrix-related genes in radiation enteritis compared with healthy ileum. cDNA array analysis revealed a strong induction of genes coding for collagens I, III, IV, VI, and VIII, SPARC, and tenascin-C, extracellular-matrix degrading enzymes (MMP-1, -2, -3, -14, -18+19), and metalloproteinase inhibitors (TIMP-1, -2, plasminogen activator inhibitor-1) in radiation enteritis. This increase was correlated with the degree of infiltration of the mucosa by inflammatory cells, and the presence of differentiated mesenchymal cells in the submucosa and muscularis propria. Despite the fact that expression of collagens, MMPs, and TIMPs simultaneously increase, quantification of net collagen deposition shows an overall accumulation of collagen. Our results indicate that late radiation enteritis tissues are subjected to active process of fibrogenesis as well as fibrolysis, with a balance toward fibrogenesis. This demonstrates that established fibrotic tissue is not scarred fixed tissue but is subjected to a dynamic remodeling process.     

Intrinsic fibroblast-mediated remodeling of damaged collagenous matrices in vivo

Numerous studies have examined wound healing and tissue repair after a complete tissue rupture and reported provisional matrix and scar tissue formation in the injury gap. The initial phases of the repair are largely mediated by the coagulation response and a principally extrinsic inflammatory response followed by type III collagen deposition to form scar tissue that may be later remodeled. In this study, we examine subfailure (Grade II sprain) damage to collagenous matrices in which no gross tissue gap is present and a localized concentration of provisional matrix or scar tissue does not form. This results in extracellular matrix remodeling that relies heavily upon type I collagen, and associated proteoglycans, and less heavily on type III scar tissue collagen. For instance, following subfailure tissue damage, collagen I and III expression was suppressed after 1 day, but by day 7 expression of both genes was significantly increased over controls, with collagen I expression significantly larger than type III expression. Concurrent with increased collagen expression were significantly increased expression of the collagen fibrillogenesis supporting proteoglycans fibromodulin, lumican, decorin, the large aggregating proteoglycan versican, and proteases cathepsin K and L. Interestingly, this remodeling process appears intrinsic with little or no inflammation response as damaged tissues show no changes in macrophage or neutrophils levels following injury and expression of the inflammatory markers, tumor necrosis factor-alpha and tartrate-resistant acid phosphatase were unchanged. Hence, since inflammation plays a large role in wound healing by inducing cell migration and proliferation, and controlling extracellular matrix scar formation, its absence leaves fibroblasts to principally direct tissue remodeling. Therefore, following a Grade II subfailure injury to the collagen matrix, we conclude that tissue remodeling is fibroblast-mediated and occurs without scar tissue formation, but instead with type I collagen fibrillogenesis to repair the tissue. As such, this system provides unique insight into acute tissue damage and offers a potentially powerful model to examine fibroblast behavior.     

Free radical involvement in hypertrophic scar formation

Hypertrophic scarring following thermal injury has become a major problem in Hong Kong. There is evidence that immunological and biochemical changes are associated with thermal injury, including pyridinoline crosslinks which are present in large quantities in hypertrophic scar, but the primary cause of hypertrophic scar formation still remains to be established. It has been reported that free radicals are assosciated with the formation of pyridinoline. In this study, attempts have been made to elucidate the involvement of free radicals in hypertrophic scar formation after thermal injury by determining the concentrations of Complement, free iron and pyridinoline crosslinks in collagen fibres. The results showed that the Complement activation product, C3d, was increased in the first week (i.e., day 7) postburn, indicating an acute inflammatory response. Free radicals, reported to be associated with the formation of pyridinoline crosslinks, and free iron content, were also found to have higher concentration in hypertrophic scar than in normal skin. The data suggest the involvement of free radical in hypertrophic scar formation. The observed increase in serum C3d concentration in about the first week indicates an acute inflammatory response to thermal injury. Both C3d and free iron concentrations (in vitro) are found higher in hypertrophic scar than in normal skin may suggest their roles in the generation of free radicals.     

CXC chemokines and their receptors: a case for a significant biological role in cutaneous wound healing

Wound healing requires a complex series of reactions and interactions among cells and their mediators, resulting in an overlapping series of events including coagulation, inflammation, epithelialization, formation of granulation tissue, matrix and scar formation. Cytokines and chemokines promote inflammation, angiogenesis, facilitate the passage of leukocytes from circulation into the tissue, and contribute to the regulation of epithelialization. They integrate inflammatory events and reparative processes that are important for modulating wound healing. Thus both cytokines and chemokines are important targets for therapeutic intervention. The chemokine-mediated regulation of angiogenesis is highly sophisticated, fine tuned, and involves pro-angiogenic chemokines, including CXCL1-3, 5-8 and their receptors, CXCR1 and CXCR2. CXCL1 and CXCR2 are expressed in normal human epidermis and are further induced during the wound healing process of human burn wounds, especially during the inflammatory, epithelialization and angiogenic processes. Human skin explant studies also show CXCR2 is expressed in wounded keratinocytes and Th/1/Th2 cytokine modulation of CXCR2 expression correlates with proliferation of epidermal keratinocytes. Murine excision wound healing, chemical burn wounds and skin organ culture systems are valuable models for examining the role of inflammatory cytokines and chemokines in wound healing.     

Matrix vesicles in aging cartilage.

The calcification process that occurs in aging has been studied with the electron microscope in costal and tracheal cartilage of rats and in human costal cartilage. In these tissues, the early stage of the calcification process is induced and regulated by matrix vesicles in the same way as it occurs in epiphyseal cartilage, bone, and dentine. However, the spreading of inorganic substance from vesicles into the surrounding matrix is frequently impaired in aged cartilage, either because of a too low concentration of calcium ions, or because the structure of the cartilage matrix is not suitable for inorganic substance deposition. This shows that matrix vesicles have a calcium affinity and calcium-binding potentiality greater than that of other components of the cartilage matrix. Most matrix vesicles are produced by "Verd?mmerung der Zellen." This degenerative process of the chondrocytes leads also to the formation of pericellular halos consisting of aggregates of amorphous substance and thin filaments. Part of the material that forms these aggregates seems to be produced by disruption of matrix vesicles. Within this disruptive material, thick collagen fibrils can be formed. Moreover, this material seems capable of inducing calcification. These findings suggest that matrix vesicles, by releasing their content into the matrix, can be involved in some way in collagen formation, and that the released material maintains the calcium affinity and calcium-binding property it has within the vesicles.     

Cellular and molecular pathways that lead to progression and regression of renal fibrogenesis

Renal fibrosis is a common consequence and often a central feature of all the progressive renal diseases that lead to end-stage renal failure. In comparison to wound healing, during kidney fibrosis the length of the post-inflammatory phase often exceeds and continues unchecked resulting in scar formation. Infiltrating immune cells and a heterogeneous colony of interstitial cells derived from a variety of cellular origins such as resident mesenchymal cells, tubular epithelial cells, circulating fibrocytes, and bone marrow derived stem cells, communicate with each other and with inflamed and surviving parenchymal cells via a network of cytokines and adhesion molecules to populate the renal tubulointerstitial space during early fibrogenesis. Such fibroblasts subsequently secrete abundant extracellular matrix to achieve architectural remodeling in parallel with functional deterioration. Renal fibrosis is a dominant determinant of the clinical outcome of patients and yet for the most part, current therapies are ineffective or only marginally effective. This review highlights recent advances in our understanding of the cellular and molecular events leading to the progression of renal fibrosis.     

Focal adhesion kinase links mechanical force to skin fibrosis via inflammatory signaling

Exuberant fibroproliferation is a common complication after injury for reasons that are not well understood. One key component of wound repair that is often overlooked is mechanical force, which regulates cell-matrix interactions through intracellular focal adhesion components, including focal adhesion kinase (FAK). Here we report that FAK is activated after cutaneous injury and that this process is potentiated by mechanical loading. Fibroblast-specific FAK knockout mice have substantially less inflammation and fibrosis than control mice in a model of hypertrophic scar formation. We show that FAK acts through extracellular-related kinase (ERK) to mechanically trigger the secretion of monocyte chemoattractant protein-1 (MCP-1, also known as CCL2), a potent chemokine that is linked to human fibrotic disorders. Similarly, MCP-1 knockout mice form minimal scars, indicating that inflammatory chemokine pathways are a major mechanism by which FAK mechanotransduction induces fibrosis. Small-molecule inhibition of FAK blocks these effects in human cells and reduces scar formation in vivo through attenuated MCP-1 signaling and inflammatory cell recruitment. These findings collectively indicate that physical force regulates fibrosis through inflammatory FAK-ERK-MCP-1 pathways and that molecular strategies targeting FAK can effectively uncouple mechanical force from pathologic scar formation.     

微小RNA miR-21在皮肤创伤愈合中的研究进展

微小RNA是一类真核细胞中广泛存在的内源性转录后调控分子,其在细胞的增殖、分化、凋亡、迁移等过程中发挥了重要的调控作用。皮肤创伤修复涉及复杂的细胞与分子的相互作用网络。近年来研究表明micro RNAs在皮肤创伤修复中发挥调控作用,引人关注。miR-21作为重要的癌基因是目前研究的最多的miRNAs分子之一,其在皮肤创伤修复中的作用研究也越来越受到重视。研究表明miR-21参与了细胞增殖与迁移、炎症反应、血管生成和细胞外基质合成等重要修复相关事件的调控。因此,阐明miR-21分子在正常皮肤创伤愈合中的作用,厘清miR-21表达失调在修复不足和修复过度中的功能,将深化我们对于皮肤创伤愈合基本理论的认识,并为促进创面愈合与防治修复不足和过度提供潜在的治疗靶点。本文就miR-21分子在正常皮肤创伤修复、慢性难愈性创面和增生性瘢痕中作用的研究进展进行综述展望。     

Scarless fetal wound healing: a basic science review

SUMMARY: Scar formation is a major medical problem that can have devastating consequences for patients. The adverse physiological and psychological effects of scars are broad, and there are currently no reliable treatments to prevent scarring. In contrast to adult wounds, early gestation fetal skin wounds repair rapidly and in the absence of scar formation. Despite extensive investigation, the exact mechanisms of scarless fetal wound healing remain largely unknown. For some time, it has been known that significant differences exist among the extracellular matrix, inflammatory response, cellular mediators, and gene expression profiles of fetal and postnatal wounds. These differences may have important implications in scarless wound repair.     

Pathogenesis of liver fibrosis: role of oxidative stress

In the liver, the progressive accumulation of connective tissue, a complex and dynamic process termed fibrosis, represents a very frequent event following a repeated or chronic insult of sufficient intensity to trigger a "wound healing"-like reaction. The fibrotic process recognises the involvement of various cells and different factors in bringing about an excessive fibrogenesis with disruption of intercellular contacts and interactions and of extracellular matrix composition. However, Kupffer cells, together with recruited mononuclear cells, and hepatic stellate cells are by far the key-players in liver fibrosis. Their cross-talk is triggered and favoured by a series of chemical mediators, with a prominent role played by the transforming growth factor beta. Both expression and synthesis of this inflammatory and pro-fibrogenic cytokine are mainly modulated through redox-sensitive reactions. Further, involvement of reactive oxygen species and lipid peroxidation products can be clearly demonstrated in other fundamental events of hepatic fibrogenesis, like activation and effects of stellate cells, expression of metalloproteinases and of their specific inhibitors. The important outcome of such findings as regards the pathogenesis of liver fibrosis derives from the observation of a consistent and marked oxidative stress condition in many if not all chronic disease processes affecting hepatic tissue. Hence, reactive oxidant species likely contribute to both onset and progression of fibrosis as induced by alcohol, viruses, iron or copper overload, cholestasis, hepatic blood congestion.     

Endothelial cells enhance adipose mesenchymal stromal cell‐mediated matrix contraction via ALK receptors and reduced follistatin: Potential role of endothelial cells in skin fibrosis

Abnormal cutaneous wound healing can lead to formation of fibrotic hypertrophic scars. Although several clinical risk factors have been described, the cross‐talk between different cell types resulting in hypertrophic scar formation is still poorly understood. The aim of this in vitro study was to investigate whether endothelial cells (EC) may play a role in skin fibrosis, for example, hypertrophic scar formation after full‐thickness skin trauma. Using a collagen/elastin matrix, we developed an in vitro fibrosis model to study the interaction between EC and dermal fibroblasts or adipose tissue‐derived mesenchymal stromal cells (ASC). Tissue equivalents containing dermal fibroblasts and EC displayed a normal phenotype. In contrast, tissue equivalents containing ASC and EC displayed a fibrotic phenotype indicated by contraction of the matrix, higher gene expression of ACTA2, COL1A, COL3A, and less secretion of follistatin. The contraction was in part mediated via the TGF‐β pathway, as both inhibition of the ALK4/5/7 receptors and the addition of recombinant follistatin resulted in decreased matrix contraction (75 ± 11% and 24 ± 8%, respectively). In conclusion, our study shows that EC may play a critical role in fibrotic events, as seen in hypertrophic scars, by stimulating ASC‐mediated matrix contraction via regulation of fibrosis‐related proteins.     

The extracellular matrix of lip wounds in fetal, neonatal and adult mice.

Wound healing in the fetus occurs rapidly, by a regenerative process and without an inflammatory response, resulting in complete restitution of normal tissue function. By contrast, in the adult, wounds heal with scar formation, which may impair function and inhibit further growth. The cellular mechanisms underlying these differing forms of wound healing are unknown but the extracellular matrix (ECM), through its effects on cell function, may play a key role. We have studied the ECM in upper lip wounds of adult, neonatal and fetal mice at days 14, 16 and 18 of gestation. The spatial and temporal distribution of collagen types I, III, IV, V and VI, fibronectin, tenascin, laminin, chondroitin and heparan sulphates were examined immunohistochemically. Results from the fetal groups were essentially similar whilst there were distinct differences between fetus, neonate and adult. Fibronectin was present at the surface of the wound in all groups at 1 h post-wounding. Tenascin was also present at the wound surface but the time at which it was first present differed between fetus (1 h), neonate (12 h) and adult (24 h). The time of first appearance paralleled the rate of wound healing which was most rapid in the fetus and slowest in the adult. Tenascin inhibits the cell adhesion effect of fibronectin and during development the appearance of tenascin correlates with the initiation of cell migration. During wound healing the appearance of tenascin preceded cell migration and the rapid closure of fetal wounds may be due to the early appearance of tenascin in the wound. Collagen types I, III, IV, V and VI were present in all three wound groups but the timing and pattern of collagen deposition differed, with restoration of the normal collagen pattern in the fetus and a scar pattern in the adult. This confirms that lack of scarring in fetal wounds is due to the organisation of collagen within the wound and not simply lack of collagen formation. The distribution of chondroitin sulphate differed between normal fetal and adult tissues and between fetal and adult wounds. Its presence in the fetal wound may alter collagen fibril formation. No inflammatory response was seen in the fetal wounds. The differences in the ECM of fetal and adult wounds suggests that it may be possible to alter the adult wound so that it heals by a fetal-like process without scar formation, loss of tissue function or restriction of growth.     

Cholesterol and cholate components of an atherogenic diet induce distinct stages of hepatic inflammatory gene expression

Atherosclerosis in inbred mouse strains has been widely studied by using an atherogenic (Ath) diet containing cholesterol, cholic acid, and fat, but the effect of these components on gene expression has not been systematically examined. We employed DNA microarrays to interrogate gene expression levels in liver of C57BL/6J mice fed the following five diets: mouse chow, the Ath diet, or modified versions of the Ath diet in which either cholesterol, cholate, or fat were omitted. Dietary cholesterol and cholate produced discrete gene expression patterns. Cholesterol was required for induction of genes involved in acute inflammation, including three genes of the serum amyloid A family, three major histocompatibility class II antigen genes, and various cytokine-related genes. In contrast, cholate induced expression of genes involved in extracellular matrix deposition in hepatic fibrosis, including five collagen family members, collagen-interacting proteins, and connective tissue growth factor. The gene expression findings were confirmed by biochemical measurements showing that cholesterol was required for elevation of circulating serum amyloid A, and cholate was required for accumulation of collagen in the liver. The possibility that these gene expression changes are relevant to atherogenesis in C57BL/6J mice was supported by the observation that the closely related, yet atherosclerosis-resistant, C57BL/6ByJ strain was largely resistant to dietary induction of the inflammatory and fibrotic response genes. These results establish that cholesterol and cholate components of the Ath diet have distinct proatherogenic effects on gene expression and suggest a strategy to study the contribution of acute inflammatory response and fibrogenesis independently through dietary manipulation.     

Mechanisms of cadmium-mediated acute hepatotoxicity

The mechanism of cadmium-mediated acute hepatotoxicity has been the subject of numerous investigations and although some uncertainties persist, sufficient evidence has emerged to provide a reasonable account of the toxic process. Acute hepatotoxicity involves two pathways, one for the initial injury produced by direct effects of cadmium and the other for the subsequent injury produced by inflammation. Primary injury appears to be caused by the binding of Cd2+ to sulfhydryl groups on critical molecules in mitochondria. Thiol group inactivation causes oxidative stress, the mitochondrial permeability transition, and mitochondrial dysfunction. Although cadmium may injure hepatocytes directly, there are compelling reasons to believe that hepatocellular injury is produced in vivo as the result of ischemia caused by damage to endothelial cells. Secondary injury from acute cadmium exposure is thought to occur from the activation of Kupffer cells and a cascade of events involving several types of liver cells and a large number of inflammatory and cytotoxic mediators. In this regard, it is clear that Kupffer cell activation and neutrophil infiltration are important events in the toxic process, and the involvement of proinflammatory cytokines and chemokines has also been implicated. The precise roles of the soluble mediators of inflammation warrant further investigation.     

New perspectives in the differentiation of bone-forming cells.

Bone formation comprises a complex but ordered sequence of events which involves the proliferation and differentiation of chondrogenic and osteoblastic precursor cells ultimately leading to the formation of a calcified extracellular matrix. This process can be observed in vivo but under these conditions is difficult to study at the molecular level. A number of in vitro models have been developed which recapitulate discrete elements of this process. Using these models, detailed information has been obtained regarding the differentiation of bone forming cells and the molecular biology of the mineralization process. It has been shown that, in vitro, osteoblastic precursor cells can form a mineralized matrix similar to that seen in vivo. This calcification process was shown to consist of three interdependent phases: proliferation, matrix maturation and mineralization. Each of these phases was characterized by the expression of particular genes. Osteoblast precursors have been cloned and consequently shown to be able to differentiate in vitro into a number of other mesenchymal cells, supporting the theory that osteoblasts are derived from multipotent mesenchymal cells. It is possible that markers derived from these models could be used in the future to extend our knowledge of bone formation in vivo.     

Signaling Pathways in Reactive Astrocytes,a Genetic Perspective

Reactive astrocytes are associated with a vast array of central nervous system (CNS) pathologies. The activation of astrocytes is characterized by changes in their molecular and morphological features, and depending on the type of damage can also be accompanied by inflammatory responses, neuronal damage, and in severe cases, scar formation. Although reactive astrogliosis is the normal physiological response essential for containing damage, it can also have detrimental effects on neuronal survival and axon regeneration, particularly in neurodegenerative diseases. It is believed that progressive changes in astrocytes as they become reactive are finely regulated by complex intercellular and intracellular signaling mechanisms. However, these have yet to be sorted out. Much has been learned from gain-of-function approaches in vivo and culture paradigms, but in most cases, loss-of-function genetic studies, which are a critical complementary approach, have been lacking. Understanding which signaling pathways are required to control different aspects of astrogliosis will be necessary for designing therapeutic strategies to improve their beneficial effects and limit their detrimental ones in CNS pathologies. In this article, we review recent advances in the mechanisms underlying the regulation of aspects of astrogliosis, with the main focus on the signaling pathways that have been studied using loss-of-function genetic mouse models.