Chaperonin Containing T-Complex Polypeptide Subunit Eta (CCT-eta) Is a Specific Regulator of Fibroblast Motility and Contractility

Integumentary wounds in mammalian fetuses heal without scar; this scarless wound healing is intrinsic to fetal tissues and is notable for absence of the contraction seen in postnatal (adult) wounds. The precise molecular signals determining the scarless phenotype remain unclear. We have previously reported that the eta subunit of the chaperonin containing T-complex polypeptide (CCT-eta) is specifically reduced in healing fetal wounds in a rabbit model. In this study, we examine the role of CCT-eta in fibroblast motility and contractility, properties essential to wound healing and scar formation. We demonstrate that CCT-eta (but not CCT-beta) is underexpressed in fetal fibroblasts compared to adult fibroblasts. An in vitro wound healing assay demonstrated that adult fibroblasts showed increased cell migration in response to epidermal growth factor (EGF) and platelet derived growth factor (PDGF) stimulation, whereas fetal fibroblasts were unresponsive. Downregulation of CCT-eta in adult fibroblasts with short inhibitory RNA (siRNA) reduced cellular motility, both basal and growth factor-induced; in contrast, siRNA against CCT-beta had no such effect. Adult fibroblasts were more inherently contractile than fetal fibroblasts by cellular traction force microscopy; this contractility was increased by treatment with EGF and PDGF. CCT-eta siRNA inhibited the PDGF-induction of adult fibroblast contractility, whereas CCT-beta siRNA had no such effect. In each of these instances, the effect of downregulating CCT-eta was to modulate the behavior of adult fibroblasts so as to more closely approximate the characteristics of fetal fibroblasts. We next examined the effect of CCT-eta modulation on alpha-smooth muscle actin (α-SMA) expression, a gene product well known to play a critical role in adult wound healing. Fetal fibroblasts were found to constitutively express less α-SMA than adult cells. Reduction of CCT-eta with siRNA had minimal effect on cellular beta-actin but markedly decreased α-SMA; in contrast, reduction of CCT-beta had minimal effect on either actin isoform. Direct inhibition of α-SMA with siRNA reduced both basal and growth factor-induced fibroblast motility. These results indicate that CCT-eta is a specific regulator of fibroblast motility and contractility and may be a key determinant of the scarless wound healing phenotype by means of its specific regulation of α-SMA expression.     

Diminished interleukin 6 (IL-6) production during scarless human fetal wound repair

Fetal wound healing is characterized by minimal inflammation and scarless repair. IL-6 stimulates inflammation in postnatal wound healing. We hypothesized that fetal skin has a diminished IL-6 response and that exogenous IL-6 will result in scar formation. Human adult or fetal skin was placed subcutaneously in SCID mice and incisionally wounded. Wounds were excised after 4, 12, 24 or 72 h for IL-6 mRNA quantification by RT-PCR. In other grafts, 5 microgram of IL-6 was injected at wounding and then harvested at 7 days for analysis of scar formation. IL-6 production was examined in primary cultures of human fetal or adult dermal fibroblasts incubated for 8 h with 0, 0.1, 1 or 10 ng/ml of PDGF-BB. IL-6 mRNA was detected 4 h after wounding in fetal and adult wounds, but by 12 h there was no IL-6 mRNA in the fetal wounds. Adult wounds had IL-6 mRNA persisting to 72 h. IL-6 administration to fetal wounds resulted in scar formation. Fetal fibroblasts produced less IL-6 protein and mRNA at all points examined (P<0.01 vs adult). Diminished production of inflammatory cytokines such as IL-6 may be responsible for the lack of inflammation seen during fetal wound healing. Diminished inflammation may provide a permissive environment for scarless wound healing.     

Discoidin domain receptors and their ligand, collagen, are temporally regulated in fetal rat fibroblasts in vitro

The biochemical regulation of collagen deposition during adult cutaneous wound repair is poorly understood. Likewise, how collagen is perceived and modulated in fetal scarless healing remains unknown. Recently, discoidin domain receptors-1 and 2 (DDR1 and DDR2) with tyrosine kinase activity have been identified as novel receptors for collagen. In light of these findings, it was speculated that the production of collagen receptors DDR1 and DDR2 by fetal fibroblasts may be temporally regulated to correlate with the ontogeny of embryonic scar formation. More specifically, because DDRs directly bind collagen and transmit the signals intracellularly, it was hypothesized that they may play an important role in fetal scarless healing by ultimately regulating and modulating collagen production and organization. As part of a fundamental assessment to elucidate the role of DDRs in scarless fetal wound repair, the endogenous expression of DDR1, DDR2, collagen I, and total collagen, as a function of fetal Sprague-Dawley rat skin fibroblasts of different gestational ages, representing scar-free (E16.5) periods was determined. Using explanted dermal fibroblasts of gestational days E13.5, E16.5, E18.5, and E21.5 (term gestation = 21.5 days) fetuses (n = 92), [3H]proline incorporation assay and Northern and Western blotting analysis were performed to compare the expressions of these molecules with scar-free and scar-forming stages of embryonic development. These results revealed a pattern of increasing collagen production with increasing gestational ages, whereas DDR1 expression decreased with increasing gestational age. This observation suggests that elevated levels of DDR1 may play an important role in scarless tissue regeneration by early gestation fetal fibroblasts. In contrast, DDR2 was expressed by fetal rat fibroblasts at a similar level throughout gestation. These data demonstrate for the first time the temporal expression of collagen and DDR tyrosine kinases in fetal rat fibroblasts as a function of gestational ages. Overall, these data suggest that differential temporal expression of the above-mentioned molecules during fetal skin development may play an important role in the ontogeny of scar formation. Future studies will involve the characterization of the biomolecular functions of these receptor kinases during fetal wound repair.     

胎儿早期小鼠无疤痕愈合的数据转录组学分析

已有研究显示,胎龄小于14 d的小鼠胎儿皮肤能够实现无疤痕创伤愈合。深入研究胎儿无疤痕愈合的分子机理,对于伤口护理方法优化与创伤医学的发展至关重要。利用已有数据库进行不同胎龄的小鼠皮肤组织转录组深入挖掘是常用的研究方法之一。从基因表达综合数据库（gene expression omnibus,GEO）在线数据库中寻找适合无疤痕愈合研究的基因表达谱芯片（GSE71619）,其中包含胎龄14 d（E14）、胎龄18 d（E18）和6周龄成年鼠（W6）的皮肤组织样本。分别将无疤痕愈合（E14）与疤痕愈合皮肤组（E18+W6）对比分析,筛选差异表达基因（differential genes,DEGs）,获得了4 654个DEGs（|log2 FC|≥1,P<0.05）。通过维恩图分析和基因功能注释确定了228个候选基因,并分为4个不同的Cluster。进一步重点分析了Cluster 3中涉及的基因：对于E14组,上调基因主要与促进伤口愈合的组织重塑和细胞外基质（extracellular matrix,ECM）形成功能相关。通过蛋白互作网络分析,确定了17个属于胶原家族和成纤维细胞迁移相关基因的关键基因。研究结果揭示了ECM的重塑和成纤维细胞活化在早期妊娠胎儿无疤痕愈合模式中的重要性,并筛选出可能的关键基因,为无疤痕伤口护理和创伤、医美领域提供了重要实验依据。     

Matrix metalloproteinases and the ontogeny of scarless repair: the other side of the wound healing balance

Early gestation mammalian fetuses possess the remarkable ability to heal cutaneous wounds in a scarless fashion. Over the past 20 years, scientists have been working to decipher the mechanisms underlying this phenomenon. Much of the research to date has focused on fetal correlates of adult wound healing that promote fibrosis and granulation tissue formation. It is important to remember, however, that wound repair consists of a balance between tissue synthesis, deposition, and degradation. Relatively little attention has been paid to this latter component of the fetal wound healing process.In this study, we examined the ontogeny of ten matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in nonwounded fetal rat skin and fibroblasts as a function of gestational age. We used a semiquantitative polymerase chain reaction protocol to analyze these important enzymes at time points that represent both the scarless and scar-forming periods of rat gestation. The enzymes evaluated were collagenase-1 (MMP-1), stromelysin-1 (MMP-3), gelatinase A (MMP-2), gelatinase B (MMP-9), membrane-type matrix metalloproteinases (MT-MMPs) 1, 2, and 3, and TIMPs 1, 2, and 3.Results demonstrated marked increases in gene expression for MMP-1, MMP-3 and MMP-9 that correlated with the onset of scar formation in nonwounded fetal skin. Similar results were noted in terms of MMP-9 gene expression in fetal fibroblasts. These results suggest that differences in the expression of these matrix metalloproteinases may have a role in the scarless wound healing phenotype observed early in fetal rat gestation. Furthermore, our data suggest that the differential expression of gelatinase B (MMP-9) may be mediated by the fetal fibroblasts themselves.     

The Role of Interleukin-10 and Hyaluronan in Murine Fetal Fibroblast Function In Vitro: Implications for Recapitulating Fetal Regenerative Wound Healing

BackgroundMid-gestation fetal cutaneous wounds heal scarlessly and this has been attributed in part to abundant hyaluronan (HA) in the extracellular matrix (ECM) and a unique fibroblast phenotype. We recently reported a novel role for interleukin 10 (IL-10) as a regulator of HA synthesis in the fetal ECM, as well as the ability of the fetal fibroblast to produce an HA-rich pericellular matrix (PCM). We hypothesized that IL-10-mediated HA synthesis was essential to the fetal fibroblast functional phenotype and, moreover, that this phenotype could be recapitulated in adult fibroblasts via supplementation with IL-10 via an HA dependent process.Conclusions/SignificanceOur data demonstrates the functional differences between fetal and adult fibroblasts, and that IL-10 mediated HA synthesis is essential for the fetal fibroblasts'' enhanced invasion and migration properties. Moreover, IL-10 via an HA-dependent mechanism can recapitulate this aspect of the fetal phenotype in adult fibroblasts, suggesting a novel mechanism of IL-10 in regenerative wound healing.     

Scarless skin wound repair in the fetus.

The ability of a fetus to heal without scar formation depends on its gestational age at the time of injury and the size of the wound defect. In general, linear incisions heal without scar until late in gestation whereas excisional wounds heal with scar at an earlier gestational age. The profiles of fetal proteoglycans, collagens, and growth factors are different from those in adult wounds. The less-differentiated state of fetal skin is probably an important characteristic responsible for scarless repair. There is minimal inflammation in fetal wounds. Fetal wounds are characterized by high levels of hyaluronic acid and its stimulator(s) with more rapid, highly organized collagen deposition. The roles of peptide growth factors such as transforming growth factor-beta and basic fibroblast growth factor are less prominent in fetal than in adult wound healing. Platelet-derived growth factor has been detected in scarless fetal skin wounds, but its role is unknown. An understanding of scarless tissue repair has possible clinical application in the modulation of adult fibrotic diseases and abnormal scar-forming conditions.     

Scarless fetal skin wound healing update

Scar formation, a physiologic process in adult wound healing, can have devastating effects for patients; a multitude of pathologic outcomes, affecting all organ systems, stems from an amplification of this process. In contrast to adult wound repair, the early‐gestation fetal skin wound heals without scar formation, a phenomenon that appears to be intrinsic to fetal skin. An intensive research effort has focused on unraveling the mechanisms that underlie scarless fetal wound healing in an attempt to improve the quality of healing in both children and adults. Unique properties of fetal cells, extracellular matrix, cytokine profile, and gene expression contribute to this scarless repair. Despite the great increase in knowledge gained over the past decades, the precise mechanisms regulating scarless fetal healing remain unknown. Herein, we describe the current proposed mechanisms underlying fetal scarless wound healing in an effort to recapitulate the fetal phenotype in the postnatal environment. Birth Defects Research (Part C) 96:237–247, 2012. © 2012 Wiley Periodicals, Inc.     

Exosomes derived from human amniotic epithelial cells accelerate wound healing and inhibit scar formation

Wound healing is a highly orchestrated physiological process consisting of a complex events, and scarless wound healing is highly desired for the development and application in clinical medicine. Recently, we have demonstrated that human amniotic epithelial cells (hAECs) promoted wound healing and inhibited scar formation through a paracrine mechanism. However, exosomes (Exo) are one of the most important paracrine factors. Whether exosomes derived from human amniotic epithelial cells (hAECs-Exo) have positive effects on scarless wound healing have not been reported yet. In this study, we examined the role of hAECs-Exo on wound healing in a rat model. We found that hAECs, which exhibit characteristics of both embryonic and mesenchymal stem cells, have the potential to differentiate into all three germ layers. hAECs-Exo ranged from 50 to 150 nm in diameter, and positive for exosomal markers CD9, CD63, CD81, Alix, TSG101 and HLA-G. Internalization of hAECs-Exo promoted the migration and proliferation of fibroblasts. Moreover, the deposition of extracellular matrix (ECM) were partly abolished by the treatment of high concentration of hAECs-Exo (100 μg/mL), which may be through stimulating the expression of matrix metalloproteinase-1 (MMP-1). In vivo animal experiments showed that hAECs-Exo improved the skin wound healing with well-organized collagen fibers. Taken together, These findings represent that hAECs-Exo can be used as a novel hope in cell-free therapy for scarless wound healing.     

Tissue remodelling in liver diseases

Tissue remodelling is a dynamic process that occurs during fetal or adult life and involves a modification of the original organization and function of a tissue. Tissue remodelling is observed in physiological and pathological conditions such as during wound healing or in the mammary gland during the course of pregnancy. In this review we will discuss the remodelling occurring in the liver as a consequence of chronic inflammation, as observed in chronic hepatitis, or as a consequence of hepatocellular carcinoma (HCC) progression in more detail. We will consider how altered deposition and turn-over of extracellular matrix (ECM) proteins could lead to development of liver fibrosis, and how the exacerbation of fibrosis could underlie the development of cirrhosis. The involvement of inflammatory and anti-inflammatory cytokines commonly used as therapeutic agents, such as Interferon-a, is then evaluated with a particular focus on modulation of ECM proteolysis. Finally, we analyze the role of alterations of the surrounding microenvironment including ECM, growth factors, cytokines and membrane receptors for ECM ligands in the development of HCC and in its invasive behaviour.     

Fibroblast Growth Factor 2 as an Antifibrotic: Antagonism of Myofibroblast Differentiation and Suppression of Pro-Fibrotic Gene Expression

Fibrosis is a pathological condition that is characterized by the replacement of dead or damaged tissue with a nonfunctional, mechanically aberrant scar, and fibrotic pathologies account for nearly half of all deaths worldwide. The causes of fibrosis differ somewhat from tissue to tissue and pathology to pathology, but in general some of the cellular and molecular mechanisms remain constant regardless of the specific pathology in question. One of the common mechanisms underlying fibroses is the paradigm of the activated fibroblast, termed the “myofibroblast,” a differentiated mesenchymal cell with demonstrated contractile activity and a high rate of collagen deposition. Fibroblast growth factor 2 (FGF2), one of the members of the mammalian fibroblast growth factor family, is a cytokine with demonstrated antifibrotic activity in non-human animal, human, and in vitro models. FGF2 is highly pleiotropic and its receptors are present on many different cell types throughout the body, lending a great deal of variety to the potential mechanisms of FGF2 effects on fibrosis. However, recent reports demonstrate that a substantial contribution to the antifibrotic effects of FGF2 comes from the inhibitory effects of FGF2 on connective tissue fibroblasts, activated myofibroblasts, and myofibroblast progenitors. FGF2 demonstrates effects antagonistic towards fibroblast activation and towards mesenchymal transition of potential myofibroblast-forming cells, as well as promotes a gene expression paradigm more reminiscent of regenerative healing, such as that which occurs in the fetal wound healing response, than fibrotic resolution. With a better understanding of the mechanisms by which FGF2 alters the wound healing cascade and results in a shift away from scar formation and towards functional tissue regeneration, we may be able to further address the critical need of therapy for varied fibrotic pathologies across myriad tissue types.     

鹿茸角柄断面伤口无疤痕愈合过程中骨形态蛋白的表达及功能分析

为研究骨形态蛋白（bone morphogenetic proteins ,BMP）在鹿茸再生早期角柄断面伤口无疤痕愈合过程中的功能,本研究通过免疫组化技术对比分析了BMP 2 和BMP 4在正常皮肤及茸皮中的表达及分布差异,同时利用添加外源性蛋白分析了BMP 4对鹿真皮成纤维细胞和毛乳头细胞的影响。结果显示：(1)原代培养的真皮成纤维细胞表达波形蛋白阳性率几乎为100%;(2) BMP 2和BMP 4强烈表达于茸皮中新生毛囊的毛基质细胞中;(3) BMP 4可促进鹿真皮成纤维细胞向脂肪细胞转分化;(4) BMP 4可促进鹿真皮毛乳头细胞成团。以此推测BMP在鹿毛囊形成及伤口愈合过程中发挥重要作用。     

Pirfenidone attenuates the profibrotic contractile phenotype of differentiated human dermal myofibroblasts

Dysregulated wound healing after burn injury frequently results in debilitating hypertrophic scarring and contractures. Myofibroblasts, the main effector cells for dermal fibrosis, develop from normal fibroblasts via transforming growth factor beta 1 (TGF-β1). During wound healing, myofibroblasts produce extracellular matrix (ECM) proteins, modulate ECM stability, and contract the ECM using alpha smooth muscle actin (α-SMA) in contractile stress fibers. The antifibrotic pirfenidone has previously been shown to inhibit the initial differentiation of fibroblasts into myofibroblasts in vitro and act as a prophylactic measure against hypertrophic scar development in a mouse burn model. To test whether pirfenidone affects differentiated myofibroblasts, we investigated the in vitro effects of pirfenidone treatment after three to five days of stimulation with TGF-β1. In assays for morphology, protein and gene expression, and contractility, pirfenidone treatment produced significant effects. Profibrotic gene expression returned to near-normal levels, further α-SMA protein expression was prevented, and cell contraction within a stressed collagen matrix was reduced. These in vitro results promote pirfenidone as a promising antifibrotic agent to treat existing scars and healing wounds by mitigating the effects of differentiated myofibroblasts.     

CD44-dependent inflammation,fibrogenesis, and collagenolysis regulates extracellular matrix remodeling and tensile strength during cutaneous wound healing

Cutaneous wound healing consists of three main phases: inflammation, re-epithelialization, and tissue remodeling. During normal wound healing, these processes are tightly regulated to allow restoration of skin function and biomechanics. In many instances, healing leads to an excess accumulation of fibrillar collagen (the principal protein found in the extracellular matrix - ECM), and the formation of scar tissue, which has compromised biomechanics, tested using ramp to failure tests, compared to normal skin (Corr and Hart, 2013 [1]). Alterations in collagen accumulation and architecture have been attributed to the reduced tensile strength found in scar tissue (Brenda et al., 1999; Eleswarapu et al., 2011). Defining mechanisms that govern cellular functionality and ECM remodeling are vital to understanding normal versus pathological healing and developing approaches to prevent scarring. CD44 is a cell surface adhesion receptor expressed on nearly all cell types present in dermis. Although CD44 has been implicated in an array of inflammatory and fibrotic processes such as leukocyte recruitment, T-cell extravasation, and hyaluronic acid (the principal glycosaminoglycan found in the ECM) metabolism, the role of CD44 in cutaneous wound healing and scarring remains unknown. We demonstrate that in an excisional biopsy punch wound healing model, CD44-null mice have increased inflammatory and reduced fibrogenic responses during early phases of wound healing. At wound closure, CD44-null mice exhibit reduced collagen degradation leading to increased accumulation of fibrillar collagen, which persists after wound closure leading to reduced tensile strength resulting in a more severe scarring phenotype compared to WT mice. These data indicate that CD44 plays a previously unknown role in fibrillar collagen accumulation and wound healing during the injury response.     

Fibrocytes can be reprogrammed to promote tissue remodeling capacity of dermal fibroblasts

Fibroblasts play a pivotal role in wound healing process participating in both tissue fibrosis and remodeling. However, it remains unclear which factors activate such diversity of fibroblast responses and how this decision-making process is made. Previous reports have demonstrated that wound milieu stimulates the transformation of circulating precursor cells into fibrocytes. These pro-fibrogenic cells promote the collagen production by resident fibroblasts. Conversely, recruited cells with anti-fibrogenic profile that can compete with fibrocytes have not been identified. This report describes a novel transdifferentiation process of fibrocytes induced by changing culture conditions. The reprogrammed fibrocytes markedly increased cell proliferation and MMP-1 expression in dermal fibroblasts. The MMP-1 up-regulation was directly related to the number of fibrocytes that followed this cell transformation. In vitro and in vivo results have confirmed that TGF-β deprivation plays an important role in this novel fibrocyte differentiation pathway. Our findings demonstrate that, changing the fibrocyte commitment, it is possible to exponentially stimulate the tissue remodeling capacity of dermal fibroblasts. These results will open new research approaches to understand the role of cell transdifferentiation and local environment not only in the wound healing process of skin, but also in several other fibrocyte-associated diseases such as lung fibrosis, asthma, liver cirrhosis, chronic pancreatitis, and atherosclerosis.     

In vitro study of the impact of mechanical tension on the dermal fibroblast phenotype in the context of skin wound healing

Skin wound healing is finely regulated by both matrix synthesis and degradation which are governed by dermal fibroblast activity. Actually, fibroblasts synthesize numerous extracellular matrix proteins (i.e., collagens), remodeling enzymes and their inhibitors. Moreover, they differentiate into myofibroblasts and are able to develop endogenous forces at the wound site. Such forces are crucial during skin wound healing and have been widely investigated. However, few studies have focused on the effect of exogenous mechanical tension on the dermal fibroblast phenotype, which is the objective of the present paper. To this end, an exogenous, defined, cyclic and uniaxial mechanical strain was applied to fibroblasts cultured as scratch-wounded monolayers. Results showed that fibroblasts? response was characterized by both an increase in procollagen type-I and TIMP-1 synthesis, and a decrease in MMP-1 synthesis. The monitoring of scratch-wounded monolayers did not show any decrease in kinetics of the filling up when mechanical tension was applied. Additional results obtained with proliferating fibroblasts and confluent monolayer indicated that mechanical tension-induced response of fibroblasts depends on their culture conditions. In conclusion, mechanical tension leads to the differentiation of dermal fibroblasts and may increase their wound-healing capacities. So, the exogenous uniaxial and cyclic mechanical tension reported in the present study may be considered in order to improve skin wound healing.     

PAI-1 in tissue fibrosis

Fibrosis is defined as a fibroproliferative or abnormal fibroblast activation-related disease. Deregulation of wound healing leads to hyperactivation of fibroblasts and excessive accumulation of extracellular matrix (ECM) proteins in the wound area, the pathological manifestation of fibrosis. The accumulation of excessive levels of collagen in the ECM depends on two factors: an increased rate of collagen synthesis and or decreased rate of collagen degradation by cellular proteolytic activities. The urokinase/tissue type plasminogen activator (uPA/tPA) and plasmin play significant roles in the cellular proteolytic degradation of ECM proteins and the maintenance of tissue homeostasis. The activities of uPA/tPA/plasmin and plasmin-dependent MMPs rely mostly on the activity of a potent inhibitor of uPA/tPA, plasminogen activator inhibitor-1 (PAI-1). Under normal physiologic conditions, PAI-1 controls the activities of uPA/tPA/plasmin/MMP proteolytic activities and thus maintains the tissue homeostasis. During wound healing, elevated levels of PAI-1 inhibit uPA/tPA/plasmin and plasmin-dependent MMP activities, and, thus, help expedite wound healing. In contrast to this scenario, under pathologic conditions, excessive PAI-1 contributes to excessive accumulation of collagen and other ECM protein in the wound area, and thus preserves scarring. While the level of PAI-1 is significantly elevated in fibrotic tissues, lack of PAI-1 protects different organs from fibrosis in response to injury-related profibrotic signals. Thus, PAI-1 is implicated in the pathology of fibrosis in different organs including the heart, lung, kidney, liver, and skin. Paradoxically, PAI-1 deficiency promotes spontaneous cardiac-selective fibrosis. In this review, we discuss the significance of PAI-1 in the pathogenesis of fibrosis in multiple organs.