Prostaglandin E(2) inhibits fibroblast chemotaxis

Fibroblasts are the major source of extracellular connective tissue matrix, and the recruitment, accumulation, and stimulation of these cells are thought to play important roles in both normal healing and the development of fibrosis. Prostaglandin E(2) (PGE(2)) can inhibit this process by blocking fibroblast proliferation and collagen production. The aim of this study was to investigate the inhibitory effect of PGE(2) on human plasma fibronectin (hFN)- and bovine bronchial epithelial cell-conditioned medium (BBEC-CM)-induced chemotaxis of human fetal lung fibroblasts (HFL1). Using the Boyden blind well chamber technique, PGE(2) (10(-7) M) inhibited chemotaxis to hFN 40.8 +/- 5.3% (P < 0.05) and to BBEC-CM 49.7 +/- 11.7% (P < 0.05). Checkerboard analysis demonstrated inhibition of both chemotaxis and chemokinesis. The effect of PGE(2) was concentration dependent, and the inhibitory effect diminished with time. Other agents that increased fibroblast cAMP levels, including isoproterenol (10(-5) M), dibutyryl cAMP (10(-5) M), and forskolin (3 x 10(-5) M) had similar effects and inhibited chemotaxis 54.1, 95.3, and 87.0%, respectively. The inhibitory effect of PGE(2) on HFL1 cell chemotaxis was inhibited by the cAMP-dependent protein kinase (PKA) inhibitor KT-5720, which suggests a cAMP-dependent effect mediated by PKA. In summary, PGE(2) appears to inhibit fibroblast chemotaxis, perhaps by modulating the rate of fibroblast migration. Such an effect may contribute to regulation of the wound healing response after injury.     

Histamine stimulates human lung fibroblast migration

Histamine is a potent mediator in allergic inflammatory processes and is released by basophils and mast cells. The aim of this study was to investigate the effect of histamine on in vitro migration of human fetal lung fibroblasts (HFL-1) to human plasma fibronectin (HFn), a chemoattractant. Using the blindwell chamber technique, histamine alone had no chemotactic activity. However, histamine augmented HFn-induced HFL-1 migration at concentrations ranging between 0 and 10^?7 M (290.6 ± 20.8%) (P < 0.05). The concentration-response was bell-shaped. The effect of histamine increased with time. The stimulatory effect of histamine on HFL-1 migration was inhibited by an H4 receptor antagonist, JNJ7777120 (10^?5 M). Histamine’s effect was also inhibited by pertussis toxin (50 ng/ml), showing that the effect was mediated by the H4 receptor. This study demonstrated that histamine has the potential to stimulate human lung fibroblast migration, and thus may contribute to regulation of wound healing and the development of fibrotic disorders of the lung.     

Cell attachment and chemotaxis can utilize the same peptide sequence of fibronectin

Fibronectin and fragments of the parent molecule are involved in cell-substrate attachment, cell migration and chemotaxis. The cell attachment sequence, Gly-Arg-Gly-Asp-Ser-Pro, was synthesized and tested for its ability to effect ligamentum nuchae fibroblast chemotaxis. This hexapeptide and fibronectin itself both caused directed cell migration, with maximal activity in the 10(-10) to 10(-9) M range. These data demonstrate that the fibronectin cell binding site and chemotaxis site share a common sequence.     

A fibrin or collagen gel assay for tissue cell chemotaxis: assessment of fibroblast chemotaxis to GRGDSP

Fibroblast chemotaxis is implicated in many physiological processes, including wound healing and morphogenesis. We present a novel assay for chemotaxis of fibroblasts (and other slow-moving tissue cells) in a direct-viewing chamber containing a physiologically relevant three-dimensional fibrin or collagen gel in which long-lasting, spatially continuous gradients have been sustained for at least 24 h, long enough for significant fibroblast migration. This combination of features is not available in any alternative assay of comparable setup simplicity. Using a putative fibroblast chemotactic factor, the fibronectin peptide GRGDSP, we measured human foreskin fibroblast alignment in the direction along the gradient, which followed a biphasic dependence on GRGDSP concentration with an optimal concentration of about 10 nM. Time-lapse video microscopy revealed that cell migration was up the soluble GRGDSP gradient, confirming positive chemotaxis to GRGDSP and rejecting the possibility of dominant haptotaxis down the soluble GRGDSP gradient, that is, up a putative gradient of integrin-mediated adhesion induced by the soluble GRGDSP gradient.     

Fibroblast adhesion results in the induction of a matrix remodeling gene expression program

Fibrosis is believed to occur through the failure to terminate the normal tissue remodeling program. Tissue repair intimately involves the ability of fibroblasts to attach to extracellular matrix (ECM), resulting in cell migration and ECM contraction. Elevated, activated adhesive signaling is a key phenotypic hallmark of fibrotic cells. The precise contribution of adhesion to tissue remodeling and repair and fibrotic responses in fibroblasts is unclear, but involves focal adhesion kinase (FAK). FAK signals downstream of integrin-mediates attachment of fibroblasts to extracellular matrix. In this report, we show that FAK is required for the expression of a cohort of mRNAs encoding ECM and matrix remodeling genes including CCN2, alpha-smooth muscle actin (SMA) and type I collagen. Adhesion of fibroblasts to fibronectin, a component of the provisional matrix deposited in the initial phases of tissue repair, also resulted in the induction of CCN2, alpha-SMA and type I collagen mRNAs. Endothelin-1 (ET-1), a key inducer of pro-fibrotic gene expression, was also induced upon fibroblast attachment to ECM, and antagonism of the ET-1 receptors significantly reduced the ability of adhesion to induce expression of CCN2, alpha-SMA and type I collagen mRNAs. These results suggest that adhesion of fibroblasts to matrix during the initial phases of tissue remodeling and repair may actively contribute to the tissue repair program through the induction of pro-fibrotic gene expression.     

Inhibition of fibroblast chemotaxis by superoxide dismutase

Superoxide radicals are known to be important mediators in chronic inflammatory and fibrotic processes, in which accumulation of fibroblasts is thought to play a major role in the pathogenetic events. The enzyme superoxide dismutase removes these radicals by a catalytic reaction. Chemotactic response of human fibroblasts and fibrosarcoma-derived cells (HT-1080) to fibroblast conditioned medium, fibronectin and platelet-derived growth factor was inhibited in a dose-dependent manner in the presence of superoxide dismutase, while random migration, cell proliferation, cell viability and synthesis of collagen and non-collagenous proteins was not altered. In contrast, phorbol myristate acetate, an inducer of superoxide generation, stimulated the chemotactic movement of fibroblasts to the attractants. Evidence for the formation of superoxide is provided by the reduction of tetrazolium salt by activated fibroblasts which could be inhibited by superoxide dismutase. Thus, it is concluded that superoxide in small amounts is involved in the mechanism of fibroblast chemotaxis. Superoxide dismutase may, therefore, reduce fibroblast migration into sites of injury or inflammation.     

Chemotactic activity of platelet alpha granule proteins for fibroblasts

At sites of blood vessel injury, platelets release numerous substances that may have biological activities influencing cellular responses. In this study we examined separately the chemotactic activity for fibroblasts of three highly purified proteins obtained from platelet alpha granules: platelet factor 4 (PF4), platelet-derived growth factor (PDGF), and beta-thromboglobulin (BTG). We observed that each of these proteins was strongly chemotactic for fibroblasts, with maximum chemotactic activity in each instance comparable to that observed with an optimal concentration of the control chemotactic protein, plasma fibronectin. Each protein was active at very low concentrations. The peak chemotactic activities of PF4, PDGF, and BTG occurred at 200 mg/ml, 30 ng/ml, and 6 ng/ml, respectively. Specificity of fibroblast chemotaxis to individual platelet proteins was provided by finding that anti-PF4 immunoglobulin blocked the chemotactic activity of PF4 without affecting the chemotactic activity of PDGF, while anti-PDGF immunoglobulin blocked the activity of PDGF but did not alter the capacity of PF4 to promote fibroblast chemotaxis. These results suggest that in vivo several alpha granule proteins released from platelets may affect wound healing by causing directed fibroblast migration.     

Smad3 mediates TGF-beta1-induced collagen gel contraction by human lung fibroblasts

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.     

Influence of alpha-tocopherol on prostacyclin synthesis by rat's arterial and myometrial tissues

Influence of alpha-tocopherol on PGI2 synthesis by rat arterial and myometrial tissues was investigated using a rat platelet antiaggregatory bioassay. Chronic administration of alpha-tocopherol to female rats (10 mg kg-1 day-1 s.c. for 14 days) significantly increased ex-vivo PGI2 synthesis by the arterial tissue from 12.7 +/- 0.3 (control, mean +/- s.e.m) to 17.2 +/- 0.4 ng PGI2 mg-1 wet tissue and by the myometrial tissue (in proestrus) from 1.1 +/- 0.07 (control) to 1.85 +/- 0.1 ng PGI2 mg-1 wet tissue (P less than 0.05, n = 6). alpha-tocopherol (5 mg kg-1 day-1 for 14 days) did not stimulate PGI2 to any significant level. Pretreatment of male rat arterial tissue with alpha-tocopherol (0.02, 0.1 or 0.2 mM) in vitro increased PGI2 synthesis in a dose-dependent manner. At a dose of 0.2 mM it increased PGI2 synthesis from 13.70 +/- 0.70 (control) to 22.6 +/- 1.4 ng PGI2 mg-1 wet tissue (P less than 0.1, n = 6). Pre-treatment of 14-day pregnant rat myometrium with alpha-tocopherol 0.2 and 0.4 mM significantly increased PGI2 synthesis from 1.2 +/- 0.06 (control) to 1.90 +/- 0.12 and 2.1 +/- 0.1 ng PGI2 mg-1 wet tissue, respectively (P less than 0.05, n = 6). The results indicate that the ability of alpha-tocopherol to stimulate PGI2 synthesis may partly contribute towards better understanding of the mechanisms that underly the protective effect of alpha-tocopherol against experimentally induced decreases in coronary flow and intravascular coagulations in some mammals. Furthermore adequate intake of alpha-tocopherol during pregnancy may enhance uterine blood flow and ensure adequate foetal nutrition.     

Inhibition of fibroblast chemotaxis by recombinant human interferon gamma and interferon alpha

Interferons have recently been recognized as potent mediators in inflammatory processes, exerting profound effects on fibroblasts. The influence of interferons gamma and alpha on the chemotactic movement of fibroblasts toward various attractants was, therefore, investigated. Normal human adult and embryonal dermal fibroblasts, fibrosarcoma-derived fibroblasts and SV40-transformed fibroblasts were tested against conditioned medium from fibroblasts, the chemotactic peptide C-140 of fibronectin, platelet-derived growth factor, and leukotriene B4 as attractants in the presence or absence of the interferons. Interferons gamma and alpha inhibited chemotaxis in a dose-dependent manner and at concentrations at least as low as 10(-2) ng/ml. Inhibition was noticeable when the cells were exposed to interferon for as short a period as 60 minutes, and the effect was not readily reversible. Inhibition occurred when the cells came from sparse or dense cultures, but when platelet-derived growth factor was the attractant and the cells had been grown at low density there was no inhibition. It is concluded that this is a specific effect, not to be wholly explained by overall increase in membrane rigidity. Inhibition of fibroblast chemotaxis by interferons may be an important regulatory mechanism during wound healing or fibrosis and metastatic spread of tumor cells.     

Morphofunctional aspects of genetic analysis of osteogenesis imperfecta in cultured skin fibroblasts]

Osteogenesis imperfecta (OI) is a disease with biochemical evidence of abnormality in collagen biosynthesis. We report here that expression of the OI phenotype extends to the level of dermal fibroblast morphology in vitro. Growth characteristics and morphology of control (n = 3) and 01 cell strains (n = 10) were compared. Our results show that (1) OI fibroblasts take longer time (16 days) than that with control cells (13 days) to reach stationary phase; (2) OI fibroblasts achieve a lower cell density (1.0 +/- 0.09) at stationary phase compared to control cells (1.47 +/- 0.1); p < 0.01; (3) cell shape (expressed as the width/length ratio) was also abnormal in OI cells: they have significantly increased ratios (p < 0.05) compared to control. These changes were associated with an increased level of fibronectin concentration and engorged cytoplasmic vesicles in dermal fibroblasts in vitro. We have reason to suspect that dysmorphology of fibroblasts may be related to aberrant collagen metabolism that leads to inadequacy of extracellular substratum, that in its turn hinders normal adhesion of cells as well as their spreading, morphology and fibronectin concentration.     

Pericytes display increased CCN2 expression upon culturing

By providing a source of α-smooth muscle actin (α-SMA)-expressing myofibroblasts, microvascular pericytes contribute to the matrix remodeling that occurs during tissue repair. However, the extent to which pericytes may contribute to the fibroblast phenotype post-repair is unknown. In this report, we test whether pericytes isolated from human placenta can in principle become fibroblast-like. Pericytes were cultured in vitro for 11 passages. The Affymetrix mRNA expression profile of passage 2 and passage 11 pericytes was compared. The expression of type I collagen, thrombospondin and fibronectin mRNAs was induced by passaging pericytes in culture. This induction of a fibroblast phenotype was paralleled by induction of connective tissue growth factor (CTGF/CCN2) and type I collagen protein expression and the fibroblast marker ASO2. These results indicate that, in principle, pericytes have the capacity to become fibroblast-like and that pericytes may contribute to the population of fibroblasts in a healed wound.     

CCN2 (connective tissue growth factor) promotes fibroblast adhesion to fibronectin

In vivo, CCN2 (connective tissue growth factor) promotes angiogenesis, osteogenesis, tissue repair, and fibrosis, through largely unknown mechanisms. In vitro, CCN2 promotes cell adhesion in a variety of systems via integrins and heparin sulfate proteoglycans (HSPGs). However, the physiological relevance of CCN2-mediated cell adhesion is unknown. Here, we find that HSPGs and the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase cascade are required for adult human dermal fibroblasts to adhere to CCN2. Endogenous CCN2 directly binds fibronectin and the fibronectin receptors integrins alpha4 beta1 and alpha5 and syndecan 4. Using Ccn2-/- mouse embryonic fibroblasts, we show that loss of endogenous CCN2 results in impaired spreading on fibronectin, delayed alpha-smooth muscle actin stress fiber formation, and reduced ERK and focal adhesion kinase phosphorylation. These results suggest that a physiological role of CCN2 is to potentiate the ability of fibroblasts to spread on fibronectin, which may be important in modulating fibroblast adhesion to the provisional matrix during tissue development and wound healing. These results are consistent with the notion that a principal function of CCN2 is to modulate receptor/ligand interactions in vivo.     

Macrophage production of fibronectin, a chemoattractant for fibroblasts

Activation of macrophages results in the production of numerous enzymes and effector molecules. One of these monokines released by macrophages can cause directed migration of connective tissue fibroblasts in vitro. Production of this macrophage-derived chemotactic factor for fibroblasts requires activation of the macrophages either in vivo or in vitro and de novo protein synthesis. The chemotactic activity in the macrophage supernatants could be removed by a fibronectin-specific affinity column and was inhibited in the presence of antibodies to fibronectin. Furthermore, chemotactic activity in the depleted macrophage supernatants could be restored by the addition of exogenous fibronectin. Fibronectin was identified in activated macrophage supernatants by an enzyme-linked immunoassay for fibronectin. From these findings it was concluded that activated macrophages release a chemoattractant for fibroblasts and that the primary chemoattractant molecule is fibronectin. The production of fibronectin by activated macrophages may thus serve as an inflammatory mediator that in addition to its other functions can recruit fibroblasts to an area of damaged tissue, where they can proliferate and form the scar tissue necessary for tissue repair. Furthermore, in chronic inflammation, the prolonged activation of macrophages may be related to the extensive fibroblast infiltration and fibrosis that can accompany these lesions.     

Biosynthesis of tissue inhibitor of metalloproteinases by human fibroblasts in culture. Stimulation by 12-O-tetradecanoylphorbol 13-acetate and interleukin 1 in parallel with collagenase

Biosynthesis of the glycoprotein tissue inhibitor of metalloproteinases (TIMP) by human fibroblasts in culture has been characterized by functional assays, immunoprecipitation, and immunocytochemistry with a monospecific antiserum. As determined by radiolabeling with [35S]methionine, immunoprecipitation, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the secreted form of TIMP had an Mr of 29,000, whereas the form associated with the cell layer had an Mr of 24,000. Unstimulated human lung fibroblasts (HFL-1) secreted TIMP at the rate of approximately 2 micrograms/10(6) cells/24 h, and normal foreskin fibroblasts (HS 27) and skin fibroblasts from a patient with Hurler's disease (GM 1391) secreted TIMP at 0.3 and 0.2 micrograms/10(6) cells/24 h, respectively. Secretion of TIMP was stimulated up to 10-fold by treating the cells with 20-100 ng/ml of 12-O-tetradecanoylphorbol 13-acetate or 10 units/ml of human interleukin 1. In the stimulated HFL-1 cells, TIMP accounted for 0.03-0.09% of the total [35S]methionine incorporated into protein, and 0.3-0.8% of the [35S]methionine in secreted protein. Although TIMP accounted for a relatively small proportion of total protein synthesis of the fibroblasts, greater than 80% of untreated and greater than 95% of stimulated fibroblasts synthesized TIMP, as determined by indirect immunofluorescence. The treatments of the human fibroblasts that increased TIMP secretion also induced synthesis and secretion of proenzyme forms of collagenase, indicating that degradative enzymes and their controlling inhibitors may be synthesized in parallel under certain conditions.     

Fibronectin, hyaluronan, and a hyaluronan binding protein contribute to increased ductus arteriosus smooth muscle cell migration.

"Intimal cushions" which develop in the late gestation lamb ductus arteriosus (DA) are characterized by smooth muscle cells migrating into a large subendothelial space. Our previous in vitro studies, comparing DA cells with those from the aorta (Ao), have shown, even in early gestation, a 10-fold increase in DA endothelial incorporation of hyaluronan into the subendothelial matrix, a 2-fold increase in smooth muscle fibronectin synthesis and, in response to endothelial conditioned medium, a 2-fold increase in chondroitin sulfate. To determine whether these extracellular matrix components may be playing a role in inducing DA smooth muscle migration, we seeded Da or Ao smooth muscle cells onto three-dimensional collagen (2.0 mg/ml) gels and assessed migration 2, 5, and 8 days later. After 8 days, significantly greater numbers of DA compared to Ao cells were found invading the gels (23.1 +/- 3.1% vs 16.2 +/- 2.3%, P less than 0.01). Addition of GRGDS peptides (0.5 mM) or antibodies against fibronectin significantly decreased migration in the DA cells, but had no effect on migration in the Ao. Addition of endothelial conditioned medium to induce smooth muscle chondroitin sulfate production had no effect on DA cell migration. Inclusion of hyaluronan in the gel (0.5-1.5 mg), however, further enhanced DA cell migration, being greatest (31.9 +/- 3.1%) at a concentration of 1 mg/ml. Hyaluronan was without effect on Ao smooth muscle cell migration. The ability of hyaluronan to promote migration in cultures of DA smooth muscle cells was blocked completely by the addition of antibodies (1:100 dilution, 1 micrograms/ml) to a cell surface hyaluronan binding protein (HABP). As well, addition of anti-HABP to cells on gels containing collagen only significantly reduced migration in the DA but not the Ao. Immunofluorescent staining revealed that in DA cells, HABP was more concentrated in lamellipodia and leading edges than in Ao cells. As well, DA smooth muscle cells synthesized greater amounts of HABP as determined by Western immunoblotting and immunoprecipitation using polyclonal antisera to HABP. Thus, our studies indicate that both increased fibronectin and HABP contribute to the enhanced migration of DA smooth muscle cells. These results, together with our previous studies showing a 10-fold increase in hyaluronan accumulation in the DA endothelial matrix, would suggest a mechanism for increased DA smooth muscle migration into the subendothelial matrix observed in vivo.     

Up-regulation of cysteinyl leukotriene 1 receptor by IL-13 enables human lung fibroblasts to respond to leukotriene C4 and produce eotaxin

Cysteinyl leukotrienes (CysLTs) play an important role in eosinophilic airway inflammation. In addition to their direct chemotactic effects on eosinophils, indirect effects have been reported. Eotaxin is a potent eosinophil-specific chemotactic factor produced mainly by fibroblasts. We investigated whether CysLTs augment eosinophilic inflammation via eotaxin production by fibroblasts. Leukotriene (LT)C(4) alone had no effect on eotaxin production by human fetal lung fibroblasts (HFL-1). However, LTC(4) stimulated eotaxin production by IL-13-treated fibroblasts, thereby indirectly inducing eosinophil sequestration. Unstimulated fibroblasts did not respond to LTC(4), but coincubation or preincubation of fibroblasts with IL-13 altered the response to LTC(4). To examine the mechanism(s) involved, the expression of CysLT1R in HFL-1 was investigated by quantitative real-time PCR and flow cytometry. Only low levels of CysLT1R mRNA and no CysLT1R protein were expressed in unstimulated HFL-1. In contrast, stimulation with IL-13 at a concentration of 10 ng/ml for 24 h significantly up-regulated both CysLT1R mRNA and protein expression in HFL-1. The synergistic effect of LTC(4) and IL-13 on eotaxin production was abolished by CysLT1R antagonists pranlukast and montelukast. These findings suggest that IL-13 up-regulates CysLT1R expression, which may contribute to the synergistic effect of LTC(4) and IL-13 on eotaxin production by lung fibroblasts. In the Th2 cytokine-rich milieu, such as that in bronchial asthma, CysLT1R expression on fibroblasts might be up-regulated, thereby allowing CysLTs to act effectively and increase eosinophilic inflammation.     

The effects of prostacyclin (PGI2) and 6-keto-PGF1 alpha on bovine plasma progesterone and LH concentrations

Injections of 1 mg PGI2 directly into the bovine corpus luteum significantly increased peripheral plasma progesterone concentrations within 5 min. Concentrations were higher in the PGI2-treated heifers than in saline-injected controls between 5 and 150 min and at 3.5, 4, 5, and 7 h post-treatment. Levels tended to remain elevated through 14 h. Saline and 6-keto-PGF1 alpha were without effect on plasma progesterone levels. The luteotrophic effect of PGI2 was not due to alterations in circulating LH concentrations. An in vitro experiment assessed the effects of either PGI2 alone or in combination with LH on progesterone production by dispersed luteal cells. Progesterone accumulation over 2 h for control, 5 ng LH, 1 microgram PGI2, 10 micrograms PGI2, and 10 micrograms PGI2 plus 5 ng LH averaged 99 +/- 42, 353 +/- 70, 152 +/- 35, 252 +/- 45, and 287 +/- 66 ng/ml (n = 4), respectively. Thus PGI2 has luteotrophic effects on the bovine CL both in vivo and in vitro.