Differential Effects of TGF-β1 on Hyaluronan Synthesis by Fetal and Adult Skin Fibroblasts: Implications for Cell Migration and Wound Healing

Fetal wound healing differs from its adult counterpart in that it is regenerative and occurs without scarring. The matrix macromolecule hyaluronan (HA) and various cytokines, including members of the TGF-β family, have been implicated in the control of scarring. We have previously reported that adult and fetal fibroblasts differ with respect to the effect of cell density on HA synthesis when cultured on plastic tissue culture dishes. Data regarding the effects of substratum and TGF-β1 on HA synthesis by these cells are presented in this communication. Our results indicate that HA synthesis by both fetal and adult fibroblasts is (a) up-regulated by culture on a collagen substratum and (b) differentially regulated by TGF-β1 in a manner which is dependent upon both substratum and cell density. TGF-β1 stimulated HA synthesis by confluent fetal fibroblasts growing on a plastic substratum, but inhibited HA synthesis on a collagen substratum; these data underscore the important role of the substratum in determining the precise effect of TGF-β1 on cell behavior. Related studies indicated that the migration of fetal and adult fibroblasts into the collagen substrata was modulated by TGF-β1 in a manner identical to its effect on HA synthesis. These observations are discussed in terms of the contribution of distinct fibroblast subpopulations to wound healing and the manner in which this is regulated by matrix and cytokines.     

IGF-1 modulation of rat cardiac fibroblast behavior and gene expression is age-dependent

The collagenous extracellular matrix (ECM) forms a stress-tolerant network that is essential for proper function of the vertebrate heart. Profound changes have been detected in the interstitial ECM concurrent with developmental and disease processes of the heart. These alterations in either the organization or accumulation of ECM components markedly affect myocardial function. Studies have shown that a number of biochemical factors, including angiotensin II, transforming growth factor-β, and insulin-like growth factors, modulate collagen expression by heart fibroblasts, however, few studies have examined the differential effects of these factors on fibroblasts from animals of different physiological backgrounds. The present studies were carried out to determine whether cardiac fibroblasts isolated from different aged animals (fetal, neonatal, and adult) have diverse responses to insulin-like growth factor-1 (IGF-1). Fibroblasts isolated from fetal, neonatal, and adult rat hearts were treated with IGF-1, and several downstream responses were measured, including collagen gel contraction, adhesion to ECM, and expression of interstitial collagen and integrins. IGF-1 affected these parameters to different degrees, depending on the age of the animal from which the fibroblasts were isolated. These experiments indicate that IGF-1 is a potent modulator of fibroblast behavior in general; however, significant differences are apparent in the responsiveness of cells to this growth factor depending on the age of the animal of origin. Future experiments will be directed at determining how the in vivo chemical and biomechanical environment affects the response of heart fibroblasts to growth factors such as IGF-1.     

Gene expression, synthesis and degradation of hyaluronan during differentiation of 3T3-L1 adipocytes

The high molecular weight glycosaminoglycan hyaluronan (HA) is an essential component of the extracellular matrix (ECM), however, the link between HA regulation and development of the adipocyte ECM, which is essential for differentiation, remains undefined. Hyaluronan synthase gene expression, HA synthetic rate and molecular weight during differentiation of 3T3-L1 pre-adipocytes were compared to undifferentiated 3T3-L1 pre-adipocytes and non-adipogenic NIH/3T3 fibroblasts. In the 3T3-L1 pre-adipocytes, the predominant genes associated with HA metabolism were found to be HA synthase-2 (Has-2) and hyaluronidase-2 (Hyal-2) demonstrating a co-regulation of expression which was stimulated by adipogenic induction consequently resulting in increased synthesis of high molecular weight HA (>10 MDa) and its simultaneous degradation. Accumulation of HA correlated positively with cell number, although synthetic rate was inversely related suggesting a regulatory feedback mechanism. Within 24h post-induction, pre-adipocytes responded with a higher HA synthetic rate and later, accumulated cytoplasmic lipid. In contrast, undifferentiated pre-adipocytes had a reduced HA synthetic rate during clonal expansion and did not accumulate lipid. HA was continuously and rapidly metabolised throughout 3T3-L1 adipogenesis, where terminal differentiation coincided with the increased generation of low molecular weight, angiogenic HA fragments, a likely prerequisite for concurrent neovascularisation of adipose tissue. This study has highlighted a relationship between HA metabolism and adipocyte differentiation, suggesting that the balance between the formation and regulation of the adipocyte extracellular matrix is finely coordinated in a growth phase-specific dependent manner.     

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.     

A pericellular hyaluronan matrix is required for the morphological maturation of cortical neurons.

BackgroundHyaluronan (HA) is a major component of the extracellular matrix (ECM) and is involved in many cellular functions. In the adult brain, HA forms macromolecular aggregates around synapses and plays important roles in neural plasticity. In contrast to the well-characterized function of HA in the adult brain, its roles in the developing brain remain largely unknown.MethodsBiochemical and histochemical analyses were performed to analyze the amount, solubility, and localization of HA in the developing mouse brain. By combining in utero labeling, cell isolation, and in vitro cultures, we examined the expression of hyaluronan synthase (HAS) and morphological maturation of cortical neurons.ResultsThe amount of HA increased during perinatal development and decreased in the adult. HA existed as a soluble form in the early stages; however, its solubility markedly decreased during postnatal development. HA localized in cell-sparse regions in the embryonic stages, but was broadly distributed during the postnatal development of the cerebral cortex. Developing cortical neurons expressed both Has2 and Has3, but not Has1, suggesting the autonomous production of HA by neurons themselves. HA formed a pericellular matrix around the cell bodies and neurites of developing cortical neurons, and the inhibition of HA synthesis reduced neurite outgrowth.ConclusionThe formation of the pericellular HA matrix is essential for the proper morphological maturation of developing neurons.General significanceThis study provides new insights into the roles of hyaluronan in the brain.development.     

Terminal differentiation of nuchal ligament fibroblasts: characterization of synthetic properties and responsiveness to external stimuli

The temporal expression of elastogenesis is unique among connective tissues in that elastin production occurs primarily during late fetal and early neonatal periods and is essentially fully repressed once fiber assembly is completed. To test whether elastin synthesis in adult nuchal ligament fibroblasts is permanently repressed or whether the cells retain the ability to reinitiate production upon proper stimulation, we examined in adult ligament cells various parameters known to be involved in the regulation of elastin production. Elastin synthetic capacity, as determined by the levels of steady-state tropoelastin mRNA, of adult tissue was significantly decreased relative to fetal tissue. Likewise, fibroblasts grown from explants of adult ligament had about a fourfold decrease in elastin production and elastin-specific mRNA levels. On the other hand, adult cells were similar to fetal ligament cells in that they were sensitive to glucocorticoid stimulation and demonstrated chemotactic responsiveness to elastin peptides. Since our previous studies have shown that the extracellular matrix (ECM) plays an important role in influencing elastin phenotypic expression, fetal and adult fibroblasts were grown on slices of nonviable adult ligament to test if repression of elastin production was directed by factors in ECM of adult tissues. No change in elastin synthesis was detected with either cell type grown on adult ligament, whereas both fetal and adult cells demonstrated increased elastin production in response to contact with fetal ligament. These results suggest that adult ligament ECM does not provide a metabolic signal to shut off the elastin gene and that adult cells remain responsive to external stimuli that may reinitiate high levels of elastin synthesis.     

Fetal and adult human skin fibroblasts display intrinsic differences in contractile capacity.

One of the differences between fetal and adult skin healing is the unique ability of fetal wounds to heal without contracture and scar formation. Studies have shown that the ratio between the three isoforms of TGFbeta is different in adult and fetal wounds. Thus, we analyzed the capacity of adult and fetal human skin fibroblasts to contract collagen gels after stimulation with TGFbeta isoforms. In control medium, fetal fibroblasts had a contractile capacity similar to that of adult fibroblasts. However, the growth capacity of fetal fibroblasts was completely inhibited, in contrast to adult fibroblasts. When cells were treated with TGFbeta, fetal fibroblasts showed an inhibition of their contractile capacity whereas adult fibroblasts further contracted gels. The contractile response was similar for all isoforms of TGFbeta although TGFbeta3 always had the strongest effect. We considered that the regulation of cell contractile capacity by TGFbeta may be dependent on receptor expression for this cytokine, on myofibroblast differentiation of the cells, or in cell links with matrix. Since TGFbeta receptor analysis did not show differences in receptor affinity, we studied the expression of alpha-smooth muscle (SM) actin, a fibroblast contractile marker and of three integrins, the cell surface receptors specific of the attachment of the fibroblasts with collagen matrix. We observed that the expression of alpha-SM actin and alpha3 and beta1 integrin subunits was increased when TGFbeta was added to the medium of adult fibroblasts whereas the levels of the alpha1 and alpha2 subunits were unchanged. In contrast, fetal fibroblasts treated with TGFbeta showed a decrease of alpha1, alpha2, and beta1 integrin expression but no change in alpha3 integrin and in alpha-SM actin expression. These results indicate that intrinsic differences between fetal and adult fibroblasts might explain their opposite responses to TGFbeta stimuli. The variations in their alpha-SM actin and integrin expression patterns represent potentially important mechanisms used by fetal fibroblasts to regulate their response to cytokines, and likely contribute to the resultant differences in the quality of wound repair.     

Ryanodine receptor 1 mediated dexamethasone-induced chondrodysplasia in fetal rats

BackgroundOsteoarthritis is caused by cartilage dysplasia and has fetal origin. Prenatal dexamethasone exposure (PDE) induced chondrodysplasia in fetal rats by inhibiting transforming growth factor β (TGFβ) signaling. This study aimed to determine the effect of dexamethasone on fetal cartilage development and illustrate the underlying molecular mechanism.MethodsDexamethasone (0.2 mg/kg.d) was injected subcutaneously every morning in pregnant rats from gestational day (GD) 9 to GD21. Harvested fetal femurs and tibias at GD21 for immunofluorescence and gene expression analysis. Fetal chondrocytes were treated with dexamethasone (100, 250 and 500 nM), endoplasmic reticulum stress (ERS) inhibitor, and ryanodine receptor 1 (RYR1) antagonist for subsequent analyses.ResultsIn vivo, prenatal dexamethasone exposure (PDE) decreased the total length of the fetal cartilage, the proportion of the proliferation area and the cell density and matrix content in fetal articular cartilage. Moreover, PDE increased RYR1 expression and intracellular calcium levels and elevated the expression of ERS-related genes, while downregulated the TGFβ signaling pathway and extracellular matrix (ECM) synthesis in fetal chondrocytes. In vitro, we verified dexamethasone significantly decreased ECM synthesis through activating RYR 1 mediated-ERS.ConclusionsPDE inhibited TGFβ signaling pathway and matrix synthesis through RYR1 / intracellular calcium mediated ERS, which ultimately led to fetal dysplasia. This study confirmed the molecular mechanism of ERS involved in the developmental toxicity of dexamethasone and suggested that RYR1 may be an early intervention target for fetal-derived adult osteoarthritis.     

Transcriptional regulation of matrix metalloproteinase-1 and collagen 1A2 explains the anti-fibrotic effect exerted by proteasome inhibition in human dermal fibroblasts

Introduction  

Extracellular matrix (ECM) turnover is controlled by the synthetic rate of matrix proteins, including type I collagen, and their enzymatic degradation by matrix metalloproteinases (MMPs). Fibrosis is characterized by an unbalanced accumulation of ECM leading to organ dysfunction as observed in systemic sclerosis. We previously reported that proteasome inhibition (PI) in vitro decreases type I collagen and enhances MMP-1 production by human fibroblasts, thus favoring an antifibrotic fibroblast phenotype. These effects were dominant over the pro-fibrotic phenotype induced by transforming growth factor (TGF)-β. Here we investigate the molecular events responsible for the anti-fibrotic phenotype induced in fibroblasts by the proteasome inhibitor bortezomib.     

Extracellular matrix regulation of fibroblast function: redefining our perspective on skin aging

The dermal extracellular matrix (ECM) comprises the bulk of skin and confers strength and resiliency. In young skin, fibroblasts produce and adhere to the dermal ECM, which is composed primarily of type I collagen fibrils. Adherence allows fibroblasts to spread and exert mechanical force on the surrounding ECM. In this state, fibroblasts display a “youthful” phenotype characterized by maintenance of the composition and structural organization of the dermal ECM. During aging, fibroblast-ECM interactions become disrupted due to fragmentation of collagen fibrils. This disruption causes loss of fibroblast spreading and mechanical force, which inextricably lead to an “aged” phenotype; fibroblasts synthesize less ECM proteins and more matrix-degrading metalloproteinases. This imbalance of ECM homeostasis further drives collagen fibril fragmentation in a self-perpetuating cycle. This article summarizes age-related changes in the dermal ECM and the mechanisms by which these changes alter the interplay between fibroblasts and their extracellular matrix microenvironment that drive the aging process in human skin.     

The role of fetal and adult hepatocyte extracellular matrix in the regulation of tissue-specific gene expression in fetal and adult hepatocytes

We explored the effect of extracellular matrix (ECM) produced by fetal and adult hepatocytes on tissue-specific gene expression and proliferation of fetal and adult hepatocytes. Adult hepatocytes ECM strongly induced expression of both albumin and HNF-4 in adult hepatocytes. In contrast, fibroblast ECM reduced the expression of mRNAs for albumin and alpha-fetoprotein in fetal hepatocytes. Adult hepatocytes ECM also increased the activity of liver-specific enzymes of adult hepatocytes (DPP IV and glucose-6-phosphatase) in both fetal and adult hepatocytes, while fetal hepatocyte-derived ECM increased activity of the fetal hepatocyte enzyme GGT in fetal hepatocytes. Fibroblast ECM was inhibitory for the activity of all enzymes assayed. Removal of heparin chains from the various matrices by pretreatment of the ECM with heparinase resulted in reduction of glucose-6-phosphatase and DPP IV in adult hepatocytes. Removal of chondroitin sulfate chains from fetal hepatocyte-derived ECM resulted in loss of induction of GGT in the fetal cells. Fetal hepatocytes proliferated best on adult hepatocyte-derived ECM. Adult hepatocytes showed only modest proliferation on both fetal and adult hepatocytes ECM and their growth was inhibited by fibroblast ECM. In conclusion, adult hepatocyte ECM better supports the expression of adult genes, whereas fetal hepatocyte ECM induced expression of fetal genes. Fibroblast derived-ECM was inhibitory for both proliferation and tissue-specific gene expression in fetal and adult hepatocytes. The data support a role for heparan sulfate being the active element in adult ECM, and chondroitin sulfate being the active element in fetal ECM.     

IL-33/HMGB-1与胎鼠皮肤伤口的瘢痕形成的相关性研究

摘要 目的：探究IL-33/HMGB-1在胎鼠伤口愈合中的作用。方法：构建小鼠伤口愈合模型,并随机分组为注射PBS组、注射重组蛋白IL-33组和重组蛋白HMGB-1组。通过免疫组织化学、DAB和苏木精复染方法检测IL-33/HMGB-1的表达及定位;结合Axiovision软件计算MOMA-2阳性巨噬细胞、波形蛋白阳性成纤维细胞和血管密度;通过Masson''s三色染色评估伤口胶原蛋白的沉积情况和愈合情况。结果：E15和E18胎鼠未损伤皮肤的基底角质形成细胞核均呈阳性染色;与E15胎鼠相比,E18胎鼠皮肤中HMGB-1和IL-33的表达水平升高（P<0.05）。处理0 h-48 h,E15和E18胎鼠伤口边缘附近角质形成细胞的核染色呈降低,IL-33和HMGB-1表达水平均降低（P<0.05）。Masson三色染色结果显示,与PBS组相比,当采取200 ng或400 ng HMGB-1或IL-33处理,E15胎鼠伤口愈合形成疤痕的数量均显著增加（P<0.05）,且疤痕大小呈剂量依赖性增加（P<0.05）。创伤后7 d,与PBS组相比,HMGB-1和IL-33处理的E15胎鼠伤口和瘢痕中波形蛋白阳性成纤维细胞、MOMA-2阳性巨噬细胞的数量和PECAM阳性血管密度均显著升高（P<0.01）。结论：IL-33/HMGB-1可以促进胎鼠伤口瘢痕的形成,其可能机制包括对成纤维细胞的直接刺激,以及与伤口中血管生成和巨噬细胞募集增加有关。     

The interplay of fibroblasts,the extracellular matrix,and inflammation in scar formation

Various forms of fibrosis, comprising tissue thickening and scarring, are involved in 40% of deaths across the world. Since the discovery of scarless functional healing in fetuses prior to a certain stage of development, scientists have attempted to replicate scarless wound healing in adults with little success. While the extracellular matrix (ECM), fibroblasts, and inflammatory mediators have been historically investigated as separate branches of biology, it has become increasingly necessary to consider them as parts of a complex and tightly regulated system that becomes dysregulated in fibrosis. With this new paradigm, revisiting fetal scarless wound healing provides a unique opportunity to better understand how this highly regulated system operates mechanistically. In the following review, we navigate the four stages of wound healing (hemostasis, inflammation, repair, and remodeling) against the backdrop of adult versus fetal wound healing, while also exploring the relationships between the ECM, effector cells, and signaling molecules. We conclude by singling out recent findings that offer promising leads to alter the dynamics between the ECM, fibroblasts, and inflammation to promote scarless healing. One factor that promises to be significant is fibroblast heterogeneity and how certain fibroblast subpopulations might be predisposed to scarless healing. Altogether, reconsidering fetal wound healing by examining the interplay of the various factors contributing to fibrosis provides new research directions that will hopefully help us better understand and address fibroproliferative diseases, such as idiopathic pulmonary fibrosis, liver cirrhosis, systemic sclerosis, progressive kidney disease, and cardiovascular fibrosis.     

Suppression of atrial natriuretic peptide/natriuretic peptide receptor-A-mediated signaling upregulates angiotensin-II-induced collagen synthesis in adult cardiac fibroblasts

Cardiac hormone atrial natriuretic peptide (ANP) and its receptor natriuretic peptide receptor-A (NPR-A) system acts as an intrinsic negative regulator of abnormal extracellular matrix (ECM) remodeling in the heart. However, the underlying mechanism by which ANP/NPR-A system opposes the ECM remodeling in the diseased heart is not well understood. Here, we investigated the anti-fibrotic mechanism of ANP/NPR-A in fibrotic agonist Angiotensin- II (ANG II)-treated adult cardiac fibroblast (CF) cells. Normal and NPR-A-suppressed adult CF cells were treated with ANG II (10^?7 M) in the presence and absence of ANP (10^?8 M) for 24 h. Total collagen concentration, activity and expression of MMP-2 and MMP-9, and nuclear translocation of Nuclear factor-kappaB (NF-κB-p50) were studied. NPR-A-suppressed adult CF cells exhibited a more pronounced increase in collagen production, ROS generation, and NF-κB-p50 nuclear translocation as compared to adult CF cells treated with agonist alone. ANP co-treatment significantly reverses the agonist-induced above changes in normal adult CF cells, while it failed to reverse the agonist-induced collagen synthesis in the NPR-A-suppressed adult CF cells. The cGMP analog (8-bromo-cGMP) treatment significantly attenuated the agonist-induced collagen synthesis both in normal and NPR-A-suppressed adult cells. The results of this study suggest that ANP/NPR-A signaling system antagonizes the agonist-induced collagen synthesis via suppressing the activities of MMP-2, MMP-9, ROS generation, and NF-κB nuclear translocation mechanism.     

Constitutively activated dystrophic muscle fibroblasts show a paradoxical response to TGF-β and CTGF/CCN2

Transforming growth factor beta (TGF-beta) and connective tissue growth factor (CTGF) have been described to induce the production of extracellular matrix (ECM) proteins and have been reported to be increased in different fibrotic disorders. Skeletal muscle fibrosis is a common feature of Duchenne muscular dystrophy (DMD). The mdx mouse diaphragm is a good model for DMD since it reproduces the muscle degenerative and fibrotic changes. Fibronectin (FN) and proteoglycans (PG) are some of the ECM proteins upregulated in dystrophic conditions. In view of understanding the fibrotic process involved in DMD we have isolated fibroblasts from dystrophic mdx diaphragms. Here we report that regardless of the absence of degenerative myofibers, adult mdx diaphragm fibroblasts show increased levels of FN and condroitin/dermatan sulfate PGs synthesis. Fibroblasts isolated from non fibrotic tissue, such as 1 week old mice diaphragms or skin, do not present elevated FN levels. Furthermore, mdx fibroblast conditioned media is able to stimulate FN synthesis in control fibroblasts. Autocrine TGF-beta signaling was unaltered in mdx cells. When control fibroblasts are exposed to TGF-beta and CTGF, FN increases as expected. Paradoxically, in mdx cells it decreases in a concentration dependent manner and this decrease is not due to a downregulation of FN synthesis. According to this data we hypothesize that a pathological environment is able to reprogram fibroblasts into an activated phenotype which can be maintained through generations.     

Fibroblast biology. Signals targeting the synovial fibroblast in arthritis

Fibroblast-like cells in the synovial lining (type B lining cells), stroma and pannus tissue are targeted by many signals, such as the following: ligands binding to cell surface receptors; lipid soluble, small molecular weight mediators (eg nitric oxide [NO], prostaglandins, carbon monoxide); extracellular matrix (ECM)-cell interactions; and direct cell-cell contacts, including gap junctional intercellular communication. Joints are subjected to cyclic mechanical loading and shear forces. Adherence and mechanical forces affect fibroblasts via the ECM (including the hyaluronan fluid phase matrix) and the pericellular matrix (eg extracellular matrix metalloproteinase inducer [EMMPRIN]) matrices, thus modulating fibroblast migration, adherence, proliferation, programmed cell death (including anoikis), synthesis or degradation of ECM, and production of various cytokines and other mediators [1]. Aggressive, transformed or transfected mesenchymal cells containing proto-oncogenes can act in the absence of lymphocytes, but whether these cells represent regressed fibroblasts, chondrocytes or bone marrow stem cells is unclear.     

Fibroblast biology: Signals targeting the synovial fibroblast in arthritis

Fibroblast-like cells in the synovial lining (type B lining cells), stroma and pannus tissue are targeted by many signals, such as the following: ligands binding to cell surface receptors; lipid soluble, small molecular weight mediators (eg nitric oxide [NO], prostaglandins, carbon monoxide); extracellular matrix (ECM)-cell interactions; and direct cell-cell contacts, including gap junctional intercellular communication. Joints are subjected to cyclic mechanical loading and shear forces. Adherence and mechanical forces affect fibroblasts via the ECM (including the hyaluronan fluid phase matrix) and the pericellular matrix (eg extracellular matrix metalloproteinase inducer [EMMPRIN]) matrices, thus modulating fibroblast migration, adherence, proliferation, programmed cell death (including anoikis), synthesis or degradation of ECM, and production of various cytokines and other mediators [1]. Aggressive, transformed or transfected mesenchymal cells containing proto-oncogenes can act in the absence of lymphocytes, but whether these cells represent regressed fibroblasts, chondrocytes or bone marrow stem cells is unclear.     

Hyaluronan concentration within a 3D collagen matrix modulates matrix viscoelasticity, but not fibroblast response

The use of 3D extracellular matrix (ECM) microenvironments to deliver growth-inductive signals for tissue repair and regeneration requires an understanding of the mechanisms of cell–ECM signaling. Recently, hyaluronic acid (HA) has been incorporated in collagen matrices in an attempt to recreate tissue specific microenvironments. However, it is not clear how HA alters biophysical properties (e.g. fibril microstructure and mechanical behavior) of collagen matrices or what impact these properties have on cell behavior. The present study determined the effects of varying high molecular weight HA concentration on 1) the assembly kinetics, fibril microstructure, and viscoelastic properties of 3D type I collagen matrices and 2) the response of human dermal fibroblasts, in terms of morphology, F-actin organization, contraction, and proliferation within the matrices. Results showed increasing HA concentration up to 1 mg/ml (HA:collagen ratio of 1:2) did not significantly alter fibril microstructure, but did significantly alter viscoelastic properties, specifically decreasing shear storage modulus and increasing compressive resistance. Interestingly, varied HA concentration did not significantly affect any of the measured fibroblast behaviors. These results show that HA-induced effects on collagen matrix viscoelastic properties result primarily from modulation of the interstitial fluid with no significant change to the fibril microstructure. Furthermore, the resulting biophysical changes to the matrix are not sufficient to modulate the cell–ECM mechanical force balance or proliferation of resident fibroblasts. These results provide new insight into the mechanisms by which cells sense and respond to microenvironmental cues and the use of HA in collagen-based biomaterials for tissue engineering.     

The role of Smad2 and Smad3 in regulating homeostatic functions of fibroblasts in vitro and in adult mice

The heart contains an abundant fibroblast population that may play a role in homeostasis, by maintaining the extracellular matrix (ECM) network, by regulating electrical impulse conduction, and by supporting survival and function of cardiomyocytes and vascular cells. Despite an explosion in our understanding of the role of fibroblasts in cardiac injury, the homeostatic functions of resident fibroblasts in adult hearts remain understudied. TGF-β-mediated signaling through the receptor-activated Smads, Smad2 and Smad3 critically regulates fibroblast function. We hypothesized that baseline expression of Smad2/3 in fibroblasts may play an important role in cardiac homeostasis. Smad2 and Smad3 were constitutively expressed in normal mouse hearts and in cardiac fibroblasts. In cultured cardiac fibroblasts, Smad2 and Smad3 played distinct roles in regulation of baseline ECM gene synthesis. Smad3 knockdown attenuated collagen I, collagen IV and fibronectin mRNA synthesis and reduced expression of the matricellular protein thrombospondin-1. Smad2 knockdown on the other hand attenuated expression of collagen V mRNA and reduced synthesis of fibronectin, periostin and versican. In vivo, inducible fibroblast-specific Smad2 knockout mice and fibroblast-specific Smad3 knockout mice had normal heart rate, preserved cardiac geometry, ventricular systolic and diastolic function, and normal myocardial structure. Fibroblast-specific Smad3, but not Smad2 loss modestly but significantly reduced collagen content. Our findings suggest that fibroblast-specific Smad3, but not Smad2, may play a role in regulation of baseline collagen synthesis in adult hearts. However, at least short term, these changes do not have any impact on homeostatic cardiac function.