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One major goal in tissue engineering is to create functional materials, mimicking scaffolds in native tissues, to modulate cell function for tissue repair. Collagen is the most abundant structural protein in human body. Though collagen I (COLI) and collagen III (COLIII) are the predominant collagen types in connective tissues and they form stable hybrid fibrils at varied ratios, cell responses to the hybrid matrices are underinvestigated. In this work, we aim to explicate the distinctive roles of COLI and COLIII in fibroblast activation. Unidirectionally aligned COLI, COLIII and COLI-COLIII hybrid nanofibrils were generated via epitaxial growth of collagen on mica. AFM analyses revealed that, with the increase of COLI/COLIII ratio, the fibril width and stiffness increased and the binding affinity of cells to the matrix decreased. A hybrid matrix was found to activate fibroblasts the most effectively, characterized by extensive cell polarization with rigid stress fiber bundles and high α-SMA expression, and by the highest-level of collagen synthesis. It is ascribed to the fine balance between biochemical and biophysical cues achieved on the hybrid matrix. Thus, matrices of aligned COLI-COLIII hybrid fibrils and their derived multifunctional composites can be good candidates of implantation scaffolds for tissue regeneration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

BACKGROUND Hypoxia induces cell apoptosis in the uterosacral ligaments of patients with pelvic organ prolapse by upregulation of hypoxia-inducible factor-1α (HIF-1α). This study aimed to investigate the effects of HIF-1α on human uterosacral ligament fibroblasts (hUSLFs) following treatment with the chemical inducer of hypoxia, cobalt chloride (CoCl2), and to explore the underlying mechanisms. MATERIAL AND METHODS Ten women who underwent hysterectomy for benign disease provided uterosacral ligament tissue for cell extraction. Following CoCl₂ treatment, cell viability of isolated and cultured hUSLFs was evaluated by the MTT assay. JC-1 fluorescence mitochondrial imaging was used to study the change in mitochondrial membrane potential. Cell apoptosis and expression of apoptosis-associated proteins and collagen type I alpha 1 (COL1A1) were measured by flow cytometry, TUNEL and Western blot, respectively. RESULTS Hypoxia increased the expression of HIF-1a and increased cell apoptosis, decreased cell viability and expression levels of COL1A1. The JC-1 assay showed that the mitochondrial membrane potential was reduced and caspase-8, and -9 inhibitors partly reduced hUSLF apoptosis. HIF-1α treatment downregulated the expression of cellular FLICE inhibitory protein (c-FLIP), decoy receptor 2 (DcR2), and the ratio of Bcl-2 to Bax, and upregulated the expression tumor necrosis factor related apoptosis-inducing ligand (TRAIL), death receptor 5 (DR5) or TRAIL-R2, Fas, Bcl-2 interacting protein 3 (BNIP3), and cytochrome C, and increased the activation of caspase-3, caspase-8, and caspase-9, all of which were reversed by knockdown of HIF-1α. CONCLUSIONS HIF-1α significantly induced apoptosis of hUSLFs through both the cell death receptor and the mitochondrial-associated apoptosis pathways.

Background and Objective: Collagen gels containing cells are commonly used in tissue engineering, wound healing, and cancer research to investigate the interplay between cells and the extracellular matrix (ECM), as changes in the density and stiffness of the microenvironment are known to play a role in many diseases or pathological conditions. In these gels, the stiffness is regularly determined using destructive methods, such as indentation and tensile tests. Certain molecules native to cells and the ECM display fluorescence upon irradiation with ultraviolet light. The objective of the present study was to investigate the feasibility of using the endogenous, or innate, fluorescence of collagen gels containing fibroblasts as an optical marker to evaluate changes in the mechanical properties of the ECM., Materials and Methods: Human foreskin fibroblasts cells at concentrations of 50,000 and 100,000 cells/ml were cultured in three-dimensional gels of type I collagen for 16 days. Fibroblast cells remodeled the ECM, contracting and increasing the stiffness of the gel. During this remodeling process, changes in mechanical properties and fluorescence were measured with an indentation test and a spectrofluorometer, respectively. Force and displacement measurements from the indentation test were used to calculate the elastic modulus of the gel. Maps of fluorescence intensity, at excitation/emission of 240-520/290-530 nm, were used to identify the wavelengths of interest., Results: Fluorescence excitation/emission maps exhibited two distinct excitation/emission bands whose intensities increased as the fibroblasts remodeled and increased the stiffness of the ECM: The 290/340 nm band ascribed to tryptophan and the 330/390 nm band ascribed to cross-links of collagen (pepsin-digestible cross-links). A Spearman correlation analysis, between the elastic modulus of the gel containing fibroblasts and the fluorescence of cross-links of collagen, resulted in R = 0.95 (P < 0.05) and R = 0.77 (P = 0.12) for 50,000 and 100,000 cells/ml, respectively., Conclusions: The endogenous fluorescence intensity ascribed to pepsin-digestible cross-links of collagen may serve as an optical marker to evaluate changes in the mechanical properties of the ECM; this is relevant to collagenous tissues for which pathological states are related to mechanical alterations, such as keratoconus in cornea and osteoarthritis in articular cartilage., (© 2016 Wiley Periodicals, Inc.)

Background: We previously showed that fibrocytes, a hematopoietic stem cell source of fibroblasts/myofibroblasts, infiltrated the colonic mucosa of a murine colitis model., Aim: We investigated whether fibrocytes were involved in the pathogenesis of Crohn's disease., Methods: Human surgical intestinal specimens were stained with anti-leukocyte-specific protein 1 and anti-collagen type-I (ColI) antibodies. Circulating fibrocytes in the human peripheral blood were quantified by fluorescence-activated cell sorting with anti-CD45 and anti-ColI antibodies. Cultured human fibrocytes were prepared by culturing peripheral CD14(+) monocytes., Results: In the specimens of patients with Crohn's disease, the fibrocyte/total leukocyte percentage was significantly increased in inflammatory lesions (22.2 %, p < 0.01) compared with that in non-affected areas of the intestine (2.5 %). Interestingly, the percentage in fibrotic lesions was similar (2.2 %, p = 0.87) to that in non-affected areas. The percentages of circulating fibrocytes/total leukocytes were significantly higher in patients with Crohn's disease than in healthy controls. Both CXC-chemokine receptor 4(+) and intercellular adhesion molecule 1(+) fibrocyte numbers were significantly increased in Crohn's disease, suggesting that circulating fibrocytes have a higher ability to infiltrate injured sites and traffic leukocytes. In cultured fibrocytes, lipopolysaccharide treatment remarkably upregulated tumor necrosis factor (TNF)-α mRNA (17.0 ± 5.7-fold) and ColI mRNA expression (12.8 ± 5.7-fold), indicating that fibrocytes stimulated by bacterial components directly augmented inflammation as well as fibrosis., Conclusions: Fibrocytes are recruited early in the inflammatory phase and likely differentiate into fibroblasts/myofibroblasts until the fibrosis phase. They may enhance inflammation by producing TNF-α and can directly augment fibrosis by producing ColI.

The anterior cruciate ligament (ACL) is often the target of knee trauma. This ligament does not heal very well, leading to joint instability. Long-term instability of the knee can lead to early arthritis and loss of function. To develop efficient strategies to stimulate posttraumatic ACL regeneration in vivo, a good healing model is needed in vitro. Such a model must remain as simple as possible, but should include key features to provide relevant answers to precise questions about the clinical problem addressed. Here, we report tissue-engineered type I collagen scaffolds developed to establish an ACL healing model in vitro and a potential ACL substitute in vivo. Such scaffolds were used to evaluate ACL cell growth, migration, and the capacity to synthesize and assemble collagen fibers for up to 40 days in vitro and up to 180 days in vivo. They were anchored with two bone plugs to allow their static stretching in culture and to facilitate their surgical implantation in knee joints. Our results have shown that living ACL fibroblasts can attach, migrate, and colonize this type of scaffold. In vitro, the cells populated the scaffolds and expressed mRNAs coding for the prolyl-4-hydroxylase, involved in collagen fibers' assembly. In vivo, acellular implants were vascularized and populated with caprine cells that migrated from the osseous insertions toward the center of the grafts. This model is a very good tool to study ACL repair and identify the factors that could accelerate its healing postsurgery., (© 2010 by the Wound Healing Society.)

Acute lung injury involving extremely immature lungs often heals without excessive fibrosis unlike later in gestation and in adults. Several factors may be involved, but fibroblast contraction of collagen has been linked to the level of wound fibrosis. To assess whether human lung fibroblasts of fetal versus adult origin differ in ability to contract collagen and define the molecular underpinnings, we performed three-dimensional collagen contraction assay, analyzed their differential mRNA profile, specifically for transforming growth factor-beta (TGF-beta) signaling pathway and extracellular matrix components, studied the cell response to TGF-beta in culture, and used two-dimensional gel electrophoresis followed by mass spectrometry to identify differences in their overall proteomes. Human lung fetal fibroblasts contracted the collagen matrix less than the adults. Smooth muscle actin expression did not differ. TGF-beta stimulation resulted in greater Smad3 phosphorylation in fetal compared with adults. mRNA and proteomic profiling reveal a number of TGF-beta pathways, ECM components, and cytoskeletal regulatory molecules are differentially expressed between the cell types. Of note is TGF-beta receptor interacting protein 1 (TRIP-1), which we show inhibits fibroblast collagen contraction and is higher in fetal than adult fibroblasts. We conclude that human lung fetal fibroblasts are less able to contract collagen than adult lung fibroblasts. The diminished ability is not due to impediment of Smad3 activation but rather, at least in part, due to their higher level of TRIP-1 expression. TRIP-1 is a novel modulator of fibroblast collagen contraction.

Several studies indicate that cancer-associated fibroblasts play a critical role in cancer cell invasion and metastasis, the hallmarks of malignancy. To better understand the mechanisms underlying such effects, we established a heterotypic model of human fibroblasts (primary colon fibroblasts and immortalized human dermal fibroblasts) in co-culture with human colon cancer cells (HCT-8/E11), using three-dimensional collagen type-I and Matrigel matrices. We report that TGF-beta is the unique and dominant factor to provide pro-invasive signals to HCT-8/E11 colon cancer cells from TGF-beta-treated human fibroblasts in three-dimensional collagen type I and Matrigel matrices. These effects are not mimicked or reversed by EGF or bFGF, and are associated with the TGF-beta-mediated induction of myofibroblast differentiation and functional markers, such as alpha-SMA, the haptotactic matrix molecule TNC, collagen type 1 maturation enzyme P4H, serine protease FAP, and myofibroblast contractility. Accordingly, TGF-beta induced a strong activation of RhoA and stress fiber formation in fibroblasts, with no impact on Rac1-GTP levels. In contrast, EGF down-regulated Rho-GTP levels in fibroblasts, giving permissive signals for Rac1 activation, fibroblast polarization, and invasion. Taken together, our data imply that TGF-beta and EGF exert invasive growth-promoting actions in human colon tumors through a differential and cumulative impact on the stromal and cancer cell compartments. Our data predict that inhibitors directed at this reciprocal molecular and cellular crosstalk will have therapeutic applications for targeting the invasive growth of human primary tumors and their metastatic spread.

Collagen contraction mediated by corneal fibroblasts (CFs) is implicated in the maintenance of corneal shape. Given that fibronectin is expressed at sites of corneal stromal wounding, we investigated the effect of fibronectin on CF-mediated collagen gel contraction. Human CFs were cultured in a three-dimensional gel of type I collagen in the absence or presence of various extracellular matrix (ECM) components. The contraction of collagen gels mediated by CFs was evaluated by measurement of changes in gel diameter. The formation of stress fibers and focal adhesions in CFs was examined by fluorescence microscopy. The abundance of paxillin, phosphorylated paxillin, integrins alpha5, beta1, and alpha2, and alpha-smooth muscle actin in CFs was examined by immunoblot analysis. Fibronectin promoted CF-mediated collagen gel contraction in a concentration- and time-dependent manner. Other ECM proteins or glycosaminoglycans did not exhibit such an effect. Fibronectin also induced cell spreading, the formation of stress fibers, and the establishment of focal adhesions containing paxillin in CFs cultured in three-dimensional collagen gels. In addition, it increased the amounts of paxillin, phosphorylated paxillin, and integrins alpha5 and beta1 in these cells. The expression of integrin alpha2 and alpha-smooth muscle actin was not affected by fibronectin, however. Furthermore, the peptide GRGDSP (an antagonist of fibronectin receptors) blocked the stimulatory effect of fibronectin on CF-mediated collagen gel contraction. These results suggest that fibronectin promoted CF-mediated collagen gel contraction in a manner dependent on the formation of stress fibers and focal adhesions, the activation of paxillin, and the up-regulation of integrin alpha5beta1. Fibronectin may therefore contribute to the maintenance of corneal shape by CFs during the healing of stromal wounds.

Enamel matrix proteins (EMP), extracted from developing porcine teeth, promote not only periodontal regeneration but also cutaneous wound healing presumably via the amelogenin fraction. Because it is unclear whether the effect of EMP can be ascribed to amelogenins, we compared EMP with recombinant amelogenin in the relaxed dermal equivalent (DE) in vitro model for early wound contraction. EMP and recombinant porcine amelogenin (rP172) at 1 mg/ml were incorporated into DEs composed of human dermal fibroblasts and a type I collagen matrix. The area reduction, as a measure of contraction, as well as fibroblast numbers and TGF-beta1 levels, were quantified over 7 days in culture in the presence of 10% foetal bovine serum. Both EMP and recombinant amelogenin increased contraction (p < 0.005) and fibroblast numbers (p < 0.005) compared with controls (acetic acid vehicle and 1mg/ml porcine serum albumin) and the positive control TGF-beta1 added at 10 ng/ml. Increased contraction with EMP and recombinant amelogenin was most pronounced after the first day of incubation and was associated with elevated (p < 0.005) TGF-beta1 levels in conditioned medium. In conclusion, the amelogenin component of EMP augmented fibroblast-driven collagen matrix remodelling, at least partially, by increasing the endogenous production of TGF-beta1. These effects of EMP/amelogenin may be beneficial for cutaneous wound healing.

Transforming growth factor-beta1 (TGF-beta1) is a key mediator in tissue repair and fibrosis. Using small interference RNA (siRNA), the role of Smad2 and Smad3 in TGF-beta stimulation of human lung fibroblast contraction of collagenous matrix and induction of alpha-SMA and the role of alpha-SMA in contraction were assessed. HFL-1 cells were transfected with Smad2, Smad3 or control-siRNA, and cultured in floating Type I collagen gels +/- -TGF-beta1. TGF-beta1 augmented gel contraction in Smad2-siRNA- and control-siRNA-treated cells, but had no effect in Smad3-siRNA-treated cells. Similarly, TGF-beta1 upregulated alpha-SMA in Smad2-siRNA- and control-siRNA-treated cells, but had no effect on Smad3-siRNA-treated cells. Alpha-SMA-siRNA-treated cells did not contact the collagen gels with or without TGF-beta1, suggesting alpha-SMA is required for gel contraction. Thus, Smad3 mediates TGF-beta1-induced contraction and alpha-SMA induction in human lung fibroblasts. Smad3, therefore, could be a target for blocking contraction of human fibrotic tissue induced by TGF-beta1.

Cardiac fibroblast (CF) proliferation and differentiation into hypersecretory myofibroblasts can lead to excessive extracellular matrix (ECM) production and cardiac fibrosis. In turn, the ECM produced can potentially activate CFs via distinct feedback mechanisms. To assess how specific ECM components influence CF activation, isolated CFs were plated on specific collagen substrates (type I, III, and VI collagens) before functional assays were carried out. The type VI collagen substrate potently induced myofibroblast differentiation but had little effect on CF proliferation. Conversely, the type I and III collagen substrates did not affect differentiation but caused significant induction of proliferation (type I, 240.7 +/- 10.3%, and type III, 271.7 +/- 21.8% of basal). Type I collagen activated ERK1/2, whereas type III collagen did not. Treatment of CFs with angiotensin II, a potent mitogen of CFs, enhanced the growth observed on types I and III collagen but not on the type VI collagen substrate. Using an in vivo model of myocardial infarction (MI), we measured changes in type VI collagen expression and myofibroblast differentiation after post-MI remodeling. Concurrent elevations in type VI collagen and myofibroblast content were evident in the infarcted myocardium 20-wk post-MI. Overall, types I and III collagen stimulate CF proliferation, whereas type VI collagen plays a potentially novel role in cardiac remodeling through facilitation of myofibroblast differentiation.

Apoptosis of fibroblasts may be key for the removal of cells following repair processes. Contraction of three-dimensional collagen gels is a model of wound healing and remodeling. Here two potent inducers of contraction, TGF-beta1 and fetal calf serum (FCS) were evaluated for their effect on fibroblast apoptosis in contracting collagen gels. Human fetal lung fibroblasts were cultured in floating type I collagen gels, exposed to TGF-beta1 or FCS, and allowed to contract for 5 days. Apoptosis was evaluated using TUNEL and confirmed by DNA content profiling. Both TGF-beta1 and serum significantly augmented collagen gel contraction. TGF-beta1 also increased apoptosis assessed by TUNEL positivity and DNA content analysis. In contrast, serum did not affect apoptosis. TGF-beta1 induction of apoptosis was associated with augmented expression of Bax, a pro-apoptotic member of the Bax/Bcl-2 family, inhibition of Bcl-2, an anti-apoptotic member of the same family, and inhibition of both cIAP-1 and XIAP, two inhibitors of the caspase cascade. Serum was associated with an increase in cIAP-1 and Bcl-2, anti-apoptotic proteins. Interestingly, serum was also associated with an apparent increase in Bax, a pro-apoptotic protein. Blockade of Smad3 with either siRNA or by using murine fibroblasts deficient in Smad3 resulted in a lack of TGF-beta induction of augmented contraction and apoptosis. Contraction induced by different factors, therefore, may be differentially associated with apoptosis, which may be related to the persistence or resolution of the fibroblasts that accumulate following injury.

A remarkable change has occurred in the thinking about epithelial-derived cancer in recent years: From almost entirely focusing on oncogenes and tumor suppressor genes has come the realization that the tumor microenvironment is a coconspirator in the carcinogenic process. Many types of stromal cells, including fibroblasts, adipocytes, macrophages, mast cells, and cells of the vascular system, are crucial contributors to epithelial carcinogenesis. Here, we focus on the fibroblast's role in cancer progression and the molecules involved in the communications between the fibroblasts and the cancer cells, including fibroblast secreted protein 1 (FSP-1 or S100A4), transforming growth factor beta (TGF-beta), the chemokine CXCL-12 (stromal derived factor 1 alpha, SDF-1alpha), type I collagen, and matrix metalloproteinase 13 (MMP-13).

The need for corneas suitable for transplantation, combined with the decreasing supply, has fueled interest in the development of a corneal replacement. In this study, a collagen-sponge-based stromal equivalent, consisting of human corneal fibroblasts cultured on a type I collagen sponge, was maintained in culture for up to 21 days and characterized with respect to mechanical properties and cellular behavior. The Young's modulus of the stromal equivalent varied from 95 to 370 Pa, and its permeability varied from 5.3 x 10(-8) - 4.2 x 10(-7) m4 N(-1) s(-1). The greatest changes occurred during the first few days in culture, but the mechanical properties continued to change during the entire 21 days. Cell traction stress, determined from sponge compaction and DNA count, decreased during the compaction process with the maximum traction value the initial value of 6.6 +/- 2.9 x 10(-3) Pacm3 cell(-1). Microarray data showed that the expression level of fibronectin, decorin sulfate, collagenase, and gelatinase A was upregulated at day 14 in the sponge. This suggested that the repair fibroblast phenotype was being expressed by the fibroblasts. Additional analysis suggested that a subpopulation of cells expressed the myofibroblast phenotype.

The extracellular matrix not only provides a structural scaffold for cells to inhabit but also forms a conduit by which mechanical information may be transmitted. Fibroblasts undergo a variety of changes when activated, including upregulating matrix metalloproteinase (MMP) activity and establishing a smooth muscle-like contractile apparatus. The relationship between MMP activity and matrix contraction has yet to be established. Here we report that inhibition of MMP activity correlates with a significant reduction in collagen gel contraction, however, force development does not change respective to MMP activity. These results suggest cellular controls of contractile forces are independent of MMP activity. Our results also raise the possibility that the material properties of the matrix dynamically change during remodeling.

TGF-beta signals through TGF-beta receptors and Smad proteins. TGF-beta also augments fibroblast-mediated collagen gel contraction, an in vitro model of connective tissue remodeling. To investigate the importance of Smad2 or Smad3 in this augmentation process, embryo-derived fibroblasts from mice lacking expression of Smad2 or Smad3 genes were cast into native type I collagen gels. Fibroblast-populated gels were then released into 0.2% FCS-DMEM alone or with recombinant human TGF-beta1, beta2, beta3, or recombinant rat PDGF-BB. Gel contraction was determined using an image analyzer. All three isoforms of TGF-beta significantly augmented contraction of collagen gels mediated by fibroblasts with genotypes of Smad2 knockout (S2KO), Smad2 wildtype (S2WT), and Smad3 wildtype (S3WT), but not Smad3 knockout (S3KO) mice. PDGF-BB augmented collagen gel contraction by all fibroblast types. These results suggest that expression of Smad3 but not Smad2 may be critical in TGF-beta augmentation of fibroblast-mediated collagen gel contraction. Thus, the Smad3 gene could be a target for blocking contraction of fibrotic tissue induced by TGF-beta., (Copyright 2003 Wiley-Liss, Inc.)

Purpose: To investigate the effect of fibronectin and tenascin on the migration of corneal fibroblasts., Setting: Department of Ophthalmology, University of Vienna, Medical School, Vienna, Austria., Methods: Using human corneal fibroblasts, a monolayer migration assay was performed to measure corneal fibroblast movement. The migration on collagen I, fibronectin, and tenascin with and without transforming growth factor (TGF)-alpha/fibroblast growth factor (FGF)-2 stimulation and the effect of soluble tenascin and fibronectin on corneal fibroblast migration on collagen-I-coated wells were investigated., Results: The cytokines TGF-alpha and FGF-2 stimulated migrational activity of corneal stromal cells in a dose-dependent manner, reaching the maximum effect at 100 ng/mL and 10 ng/mL, respectively. The migration of corneal fibroblasts on fibronectin was significantly higher (P <.05) than the migration on collagen I. Transforming growth factor-alpha and FGF-2 increased radial cell displacement independent of the provided matrix composition. Tenascin had a negative effect on corneal fibroblast adhesion/migration in this in vitro model., Conclusion: Fibronectin and tenascin influenced corneal fibroblast migration and adhesion, respectively, and may play a role in stromal cell movement during wound healing. The cytokines TGF-alpha and FGF-2 had an additive effect on corneal fibroblast migration on a fibronectin matrix.

Nerve growth factor (NGF), a polypeptide with well-known actions on neurons, is believed to play a role in the process of tissue repair. The aim of this study was to investigate the effect of NGF on human fetal lung fibroblast (HFL-1)-mediated type I collagen gel contraction and on chemotaxis of the cells with the use of the blind-well chamber technique. Neither collagen gel contraction nor the chemotaxis of HFL-1 cells was affected by NGF (100 ng/mL) alone. However, NGF significantly increased HFL-1 chemotaxis to human fibronectin (20 microg/mL) and platelet-derived growth factor-BB (PDGF-BB, 10 ng/mL), by 41.8% +/- 11.4% and 47.7% +/- 6.6%, respectively. Checkerboard analysis showed stimulation of both chemotaxis and chemokinesis. NGF appeared to affect the rate of migration. After 12 hours, control cells had migrated as much as NGF-treated cells. The effect of NGF was blocked by the tyrosine kinase receptor A inhibitor K-252a, suggesting that the biological action of NGF on fibroblast chemotaxis is mediated through this tyrosine kinase receptor. Our findings suggest that by increasing the rate at which fibroblasts migrate in response to chemoattractants, NGF can modulate the speed and intensity of a repair response and may therefore represent a valid therapeutic target for a variety of diseases.

A method to impose and measure a one dimensional strain field via confined compression of a tissue-equivalent and measure the resulting cell and collagen fibril alignment was developed Strain was determined locally by the displacement of polystyrene beads dispersed and entrapped within the network of collagen fibrils along with the cells, and it was correlated to the spatial variation of collagen network birefringence and concentration. Alignment of fibroblasts and smooth muscle cells was determined based on the long axis of elongated cells. Cell and collagen network alignment were observed normal to the direction of compression after a step strain and increased monotonically up to 50% strain. These results were independent of time after straining over 24 hr despite continued cell motility after responding instantly to the step strain with a change in alignment by deforming/convecting with the strained network. Since the time course of cell alignment followed that of strain and not stress which, due to the viscoelastic fluid-like nature of the network relaxes completely within the observation period, these results imply cell alignment in a compacting tissue-equivalent is due to fibril alignment associated with anisotropic network strain. Estimation of a contact guidance sensitivity parameter indicates that both cell types align to a greater extent than the surrounding fibrils.

Interactions of stromal and tumor cells with the extracellular matrix may regulate expression of proteases including the lysosomal proteases cathepsins B and D. In the present study, we determined whether the expression of these two proteases in human breast fibroblasts was modulated by interactions with the extracellular matrix component, collagen I. Breast fibroblasts were isolated from non-malignant breast tissue as well as from tissue surrounding malignant human breast tumors. Growth of these fibroblasts on collagen I gels affected cell morphology, but not the intracellular localization of vesicles staining for cathepsin B or D. Cathepsins B and D levels (mRNA or intracellular protein) were not affected in fibroblasts growing on collagen I gels or plastic, nor was cathepsin D secreted from these cells. In contrast, protein expression and secretion of cathepsin B, primarily procathepsin B, was induced by growth on collagen I gels. The induced secretion appeared to be mediated by integrins binding to collagen I, as inhibitory antibodies against alpha(1), alpha(2), and beta(1) integrin subunits prevented procathepsin B secretion from fibroblasts grown on collagen. In addition, procathepsin B secretion was induced when cells were plated on beta(1) integrin antibodies. To our knowledge, this is the first examination of cathepsin B and D expression and localization in human breast fibroblasts and their regulation by a matrix protein. Secretion of the cysteine protease procathepsin B from breast fibroblasts may have physiological and pathological consequences, as proteases are required for normal development and for lactation of the mammary gland, yet can also initiate and accelerate the progression of breast cancer.

Interleukin-1alpha (IL-1alpha) is strongly expressed in odontogenic keratocysts. In this study, we investigated the effects of IL-1alpha on the activation of matrix metalloproteinase-2 (MMP-2) in the fibroblasts isolated from odontogenic keratocysts. Odontogenic keratocyst fibroblasts secreted a latent form of MMP-2 (proMMP-2) spontaneously. Type I collagen induced the activation of the proMMP-2, and recombinant human IL-1alpha (rhIL-1alpha) further enhanced the type I collagen-induced activation of proMMP-2 in a dose-dependent manner. The rhIL-1alpha-induced activation of proMMP-2 was inhibited by anti-human IL-1alpha antibody. A reverse-transcription/polymerase chain-reaction (RT-PCR) and Western immunoblotting demonstrated that the expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) mRNA and protein was increased in the fibroblasts when the cells were cultured on type I collagen, and the expression was further enhanced by rhIL-1alpha. Thus, IL-1alpha may up-regulate proMMP-2 activation by increasing the expression of MT1-MMP in the fibroblasts isolated from odontogenic keratocysts synergistically with type I collagen.

The possible contributions of the angiotensin receptor subtypes 1 and 2 on the angiotensin II-induced collagen gel contraction by adult rat cardiac fibroblasts were studied using the specific angiotensin receptor type 1 and 2 antagonists telmisartan and P-186, respectively. Cardiac fibroblasts (from normal male adult rats) from passage 2 were cultured to confluency and added to a hydrated collagen gel, with or without angiotensin II, angiotensin II plus telmisartan, or angiotensin II plus P-186 in Dulbecco's Modified Eagle's Medium containing 5% fetal bovine serum for 1, 2, or 3 days. Control gels containing adult rat cardiac fibroblasts showed a significant amount of contraction after 3 days of incubation, causing a contraction to 67.9 +/- 7.1% of the area after 1 day. Angiotensin II (10(-7) M) stimulated (p < or = 0.05) the contraction of collagen mediated by cardiac fibroblasts after 1, 2, or 3 days. Telmisartan (10(-7) M) completely blocked the angiotensin II-induced collagen contraction by cardiac fibroblasts. P-186 (10(-7) M) had no effect on the angiotensin II-induced collagen contraction by cardiac fibroblasts. Addition of telmisartan and P-186 alone did not affect the collagen gel contraction by cardiac fibroblasts. Our data demonstrate that the effects of angiotensin II on the collagen gel contraction by adult rat cardiac fibroblasts are angiotensin II type 1 receptor mediated because they were abolished by the specific angiotensin II type 1 receptor antagonist telmisartan but not by the specific angiotensin II type 2 receptor antagonist P-186.