Fetal rat amniotic fluid: transforming growth factor beta and fibroblast collagen lattice contraction

BACKGROUND: In several mammalian animal models, early-gestational-age fetal wounds heal without scar, but wounds of late gestational age heal with scar. This change in wound healing phenotype can be a result of both intrinsic (i.e., cellular characteristics) and extrinsic (i.e., environmental) factors. Our question was: Does amniotic fluid (AF) influence the change from scarless to scar-forming repair in the rat? METHODS: Rat AF was investigated for its modulation of fibroblast-populated collagen lattice (FPCL) contraction and morphological changes of adult fibroblasts. AF was also assayed for transforming growth factor beta (TGF-beta) levels. Adult rat dermal fibroblasts in monolayer and incorporated into FPCLs were incubated with AF additions from gestational age 14, 16, 18, and 21 days at 10% (v/v). RESULTS: Day 14 AF significantly stimulated FPCL contraction, but AF of 16, 18, and 21 days inhibited FPCL contraction. Fluorescence histology identified microtubules and microfilaments in AF treated adult rat dermal fibroblasts. The staining pattern of microtubules in Day 14 AF-treated fibroblasts showed denser structures at the cell center. Cells incubated with Day 16 or 18 AF showed fine peripheral microtubules. A mink lung epithelial cell bioassay was used to analyze concentrations of TGF-beta in AF. TGF-beta levels were greatly elevated in Day 14 AF, but were relatively low in Day 16, 18 and 21 AF. The inhibitor of FPCL contraction from AF of Days 16, 18, and 21 was not identified. CONCLUSION: It is proposed that the robust expression of TGF-beta or cytoskeletal changes induced by Day 14 AF contributes to enhanced FPCL contraction.     

The effect of epigallocatechin-3-gallate, a constituent of green tea, on transforming growth factor-β1–stimulated wound contraction

Dermal fibrosis, or scarring, following surgical incisions, traumatic wounds and burns presents a major clinical burden. Transforming growth factor (TGF)-β1 is a major factor known to stimulate fibroblast proliferation, collagen production, and the differentiation of fibroblast to myofibroblast promoting wound contraction. Furthermore, excessive or prolonged TGF-β1 has been shown to be associated with scarring. Green tea contains high amounts of polyphenols with the major polyphenolic compound being epigallocatechin-3-gallate (EGCG). EGCG has been shown to be anti-inflammatory, anti-oxidant, and may improve wound healing and scarring, though its precise effect on TGF-β1 remains unclear. This study aimed at determining the effect of EGCG on TGF-β1 collagen contraction, gene expression and the differentiation of fibroblast to myofibroblast. EGCG appears to affect the role that TGF-β1 plays in fibroblast populated collagen gel contraction and this seems to be through both myofibroblast differentiation and connective tissue growth factor gene expression and reduces the expression of collagen type I gene regulation.     

Prostaglandin E2 differentially modulates human fetal and adult dermal fibroblast migration and contraction: implication for wound healing

Cyclooxygenase-2 is up-regulated shortly after dermal injury and it has been shown to have important activity during the repair process. Its main product in the skin, prostaglandin E2 (PGE2), modulates both inflammatory and fibrotic processes during wound healing and partially dictates the overall outcome of wound healing. PGE2 signaling has been shown to be altered during fetal wound healing. This study was designed to examine the mechanism(s) by which PGE2 regulates fibroblast migration and contraction and to determine whether these mechanisms are conserved in fetal-derived dermal fibroblasts. Fetal and adult dermal fibroblasts express all four PGE2 receptors. PGE2 inhibits fetal and adult fibroblast migration in a dose-dependent manner through the EP2/EP4-cAMP-protein kinase A pathway. However, fetal fibroblasts appear to be refractory to this effect, requiring a 10-fold higher concentration of PGE2 to achieve a similar degree of inhibition as adult fibroblasts. Inhibition of adult fibroblast migration correlated with disruption of the actin cytoskeleton. In contrast, PGE2 or a cAMP analog did not disrupt the actin cytoskeleton of fetal dermal fibroblasts. These findings were extended using a modified free-floating, fibroblast-populated collagen lattice (FPCL) contraction assay designed to measure fibroblast contraction. PGE2-inhibited FPCL contraction by adult fibroblasts, but fetal fibroblasts exhibited higher rates of FPCL contraction and a blunted response to exogenous modulation by PGE2 or a cyclase activator (forskolin). These findings indicate that fetal dermal fibroblasts are partially refractory to the effects of PGE2, a major inflammatory mediator associated with dermal wound healing. This effect may have significant and specific relevance to the scarless fetal wound-healing phenotype.     

Reversal of the wound healing deficit in diabetic rats by combined basic fibroblast growth factor and transforming growth factor-β1 therapy

Wound healing is impaired in the diabetic state because of, at least in part, low expression of growth factors. Individual growth factors can partially activate healing, yet the actual wound environment presents a dynamic continuum of multiple cellular signals. Complex interactions among growth factors and target cells can have synergistic effects, and several examples of combinatorial, in vivo activity are evident in the literature. In this study, the implantation of a combination of basic fibroblast growth factor and transforming growth factor-beta in rats induced fivefold to sevenfold increases in granulation tissue formation in comparison with implantation of each growth factor alone. Diabetes was induced in adult, male Sprague-Dawley rats with streptozotocin. Incisional wounds and sponge granulation tissue were produced in separate groups and then treated with an injection of 2 microg transforming growth factor-beta1 combined with 10 microg basic fibroblast growth factor on day 3. DNA, collagen, and protein were analyzed in granulation tissue on days 7 and 9, and biomechanical properties of incisions were tested on days 7, 14, and 21. The combination of transforming growth factor-beta1 and basic fibroblast growth factor had marked, positive effects on biochemical parameters of wound healing and reversed the tensile strength deficit of diabetic wounds. Nonradioactive in situ hybridization showed increased expression of messenger RNA for type I and III procollagen and transforming growth factor-beta1 in normal and diabetic wounds, whereas ultrastructural examination showed a marked reorganization of collagen fibrils. These findings reinforce the concept that appropriate mixtures of cytokines rather than individual cytokines may more adequately stimulate tissues in cases of impaired wound healing.     

碱性成纤维细胞生长因子拮抗转化生长因子—β1对人α1（I）胶原基因的启动激活

目的　探讨碱性成纤维细胞生长因子 (b FGF)对人α1 ( )胶原基因启动子活性的影响 ,以及与转化生长因子 - β1 (TGF- β1 )之间的相互作用 ,为防治增生性瘢痕提供依据。　方法　正常皮肤及瘢痕成纤维细胞原代、传代培养。采用 Fu GENE转染试剂 ,分别瞬间转染含人α1 ( )胶原基因 5'端序列 - 2 .5 kb与报告基因氯霉素乙酰基转移酶(CAT)的重组体 ph COL2 .5至正常皮肤及瘢痕成纤维细胞。 ELISA法测定 b FGF及 TGF- β1 作用 2 4小时后 ,转染ph COL2 .5的两种成纤维细胞的报告基因 CAT表达量。　结果　b FGF能抑制转染 ph COL2 .5重组体的正常皮肤及瘢痕成纤维细胞 CAT表达量 ,且能拮抗 TGF-β1 对转染 ph COL2 .5重组体的两种成纤维细胞 CAT表达的上调作用。与对照组相比有统计学意义 (P<0 .0 5)。　结论　正常皮肤及瘢痕成纤维细胞中 ,b FGF均能抑制人 α1 ( )胶原基因的启动转录 ,且能拮抗 TGF-β1 对人α1 ( )胶原基因启动活性的上调作用 ,b FGF抗纤维机制有望为增生性瘢痕的防治提供新思路     

Ⅱ型胶原、转化生长因子β1和碱性成纤维细胞生长因子在脊柱侧凸患者关节突软骨中分布及表达的实验研究

目的研究青少年特发性脊柱侧凸（AIS）和先天性脊柱侧凸（CS）畸形最严重部位一顶椎凸、凹侧下关节突软骨中Ⅱ型胶原、转化生长因子β1（TGF—β1）和碱性成纤维细胞生长因子（bFGF）的表达特点。方法22例AIS患者,18例CS患者作为研究对象。取两组患者的顶椎和端椎凸、凹侧的下关节突,采用苏木素-伊红（HE）染色、免疫组化和原位杂交方法观察关节突的病理改变和Ⅱ型胶原、TGF-β1及bFGF在关节突中分布的特点。将所得的免疫组化和原位杂交图像输入图像分析系统,进行半定量分析,并作统计学分析。结果Ⅱ型胶原、TGF-β1、bFGF在AIS和CS中的有基本相似的表达特点,免疫组化和原位杂交方法均显示顶椎凹侧的表达高于凸侧,差异有统计学意义（P〈0．05）;上下端椎的凸凹侧之间及凸凹侧的上下端椎之间的表达差异无统计学意义,顶椎、上下端椎各对应部位在AIS与CS之间的表达差异无统计学意义;Ⅱ型胶原在凸侧与凹侧顶椎的表达高于同侧上、下端椎,差异有统计学意义（P〈0．05）。结论AIS和CS顶椎关节突软骨呈现退变及发育不全等征象,凹侧较凸侧明显。AIS和CS中Ⅱ型胶原、TGF—β1及bFGF在顶椎凹侧表达的增高可能为脊柱畸形后异常的生物力学引起了关节突细胞间基质重建而进行代偿的结果。压应力可引起TGF—β1及bFGF的表达增高;压应力和张应力均可以引起Ⅱ型胶原的表达增高,但顶椎凹侧压应力比凸侧张应力的影响更大。     

Effects of transforming growth factor alpha and interleukin-1 on DNA synthesis, collagen synthesis, procollagen mRNA levels, and prostaglandin E2 production in cultured fetal rat calvaria

Transforming growth factor alpha (TGF-alpha) and interleukin-1 (IL-1) have been shown to affect bone metabolism in vitro by prostaglandin-dependent and PG-independent mechanisms. We assessed the effects of the combination of these two agents on [3H]thymidine (TdR) incorporation into DNA, DNA content, [3H]proline incorporation into collagenase-digestible (CDP), noncollagen protein (NCP), and PGE2 production in 21 day fetal rat calvaria cultured for 24-96 h. We also determined whether TGF-alpha plus IL-1 altered procollagen mRNA levels at 96 h. TGF-alpha, 1-30 ng/ml, produced a 41-59% increase in TdR incorporation into DNA, but the effect was partially blocked by human recombinant IL-1. At 96 h TGF-alpha alone or in combination with IL-1 significantly increased the DNA content of calvaria. At 96 h, TGF-alpha inhibited CDP labeling and the addition of IL-1 further enhanced this inhibitory effect. The enhanced inhibitory effect of TGF-alpha plus IL-1 on collagen synthesis was associated with a synergistic increase in prostaglandin accumulation in the medium. Addition of indomethacin blocked PGE2 accumulation and partially reversed the inhibitory effect of TGF-alpha alone or in combination with IL-1 on collagen synthesis. TGF-alpha decreased procollagen mRNA levels by 55%, but the combination of TGF-alpha plus IL-1 decreased procollagen mRNA levels by 82%. Our results show that TGF-alpha and IL-1, which are both produced by certain tumors as well as activated macrophages, appear to act synergistically to increase prostaglandin synthesis and inhibit collagen synthesis in vitro. Thus these agents may have a regulatory role on bone formation in vivo.     

Effects of transforming growth factor-β1 and fibroblast growth factor on DNA synthesis in growth plate chondrocytes are enhanced by insulin-like growth factor-I

The local tissue metabolism is controlled through the complex interaction between systemic and local growth factors. In recent years, an increasing number of autocrine or paracrine growth regulators have been identified in physeal cartilage. While these factors act to alter chondrocytes phenotypically and presumably are important mediators in the process of endochondral ossification, the manner in which they interact with the systemically regulated growth factor insulin-like growth factor-I is unknown. In the present study, the interactive effects of insulin-like growth factor-I with transforming growth factor-β1 or basic fibroblast growth factor were examined in short-term monolayer cultures of chick growth plate chondrocytes. [³H]thymidine incorporation was maximally stimulated 11-fold by fibroblast growth factor (10 ng/ml) and 3.5-fold by transforming growth factor-β1 following a 24-hour exposure in serum-containing cultures. The effects of transforming growth factor-β1 and fibroblast growth factor at both high and low concentrations were enhanced in a dose-dependent manner by insulin-like growth factor-I, with a 40–50% increase in DNA synthesis in the presence of 100 ng/ml of insulin-like growth factor-I. Since insulin-like growth factor-I increased [³H]thymidine incorporation after 48 hours (50% increase) but not after 24 hours of exposure, these observations represent a synergistic interaction. Total DNA in cultures treated for 5 days confirmed the modulating effect of insulin-like growth factor-I with transforming growth factor-β1 and fibroblast growth factor. The growth factors were further examined for their effects on markers of chondrocyte differentiation. While all three caused a dose-dependent inhibition of alkaline phosphatase activity, the effects of insulin-like growth factor-I were additive only to those of transforming growth factor-β1 and fibroblast growth factor. Similarly, insulin-like growth factor-I did not affect the sulfate incorporation stimulated by fibroblast growth factor or transforming growth factor-β1. Insulin-like growth factor-I had no effect on total protein synthesis after 24 hours and, although type-II collagen mRNA levels were stimulated, it had no effect on type-X collagen mRNA, as determined by quantitative in situ hybridization. Finally, insulin-like growth factor-I did not alter the dose-dependent stimulation of noncollagen protein synthesis and the inhibition of collagen synthesis caused by fibroblast growth factor and transforming growth factor-β1 in 24-hour cultures. Thus, the data suggest that insulin-like growth factor-I may have a role in augmenting the effects of other growth factors found in cartilage. Since insulin-like growth factor-I is under systemic control by growth hormones, this permits an endocrine regulation of transforming growth factor-β1 and fibroblast growth factor activity and may bring local growth factor effects under systemic control.     

Synergistic effect of transforming growth factor beta and fibroblast growth factor on DNA synthesis in chick growth plate chondrocytes

Transforming growth factor beta and fibroblast growth factor are mitogens for chick growth plate chondrocytes. TGF-beta stimulated a 3.5-fold increase, and FGF a 13.5-fold increase in the rate of thymidine incorporation after a 24 h exposure. TGF-beta and FGF were synergistic in chondrocytes, causing a 73-fold stimulation in thymidine incorporation compared with control. This synergistic response was not dependent upon the simultaneous presence of both mitogens. Sequential exposure of chondrocytes to TGF-beta and FGF in either order reproduced in large part the synergistic interaction observed when both growth factors were present simultaneously. The time required for induction of the subsequent synergistic response was brief and, in the case of TGF-beta, corresponded to the time required for [125I]TGF-beta receptor binding. EGF and PDGF were not mitogenic for chondrocytes, and neither of these factors enhanced the response of the cells to either TGF-beta or FGF. Finally, TGF-beta and FGF did not, either alone or in combination, elevate intracellular cAMP levels. These results emphasize the importance of examining growth factor effects in the context of other growth regulators. Furthermore, this specific and dramatic synergistic stimulation of thymidine incorporation may provide a useful tool in elucidating the mitogenic mechanism of the individual growth factors.     

Simvastatin inhibits transforming growth factor‐β1‐induced expression of type I collagen,CTGF, and α‐SMA in keloid fibroblasts

Simvastatin, a 3‐hydroxy‐3‐methylglutaryl coenzyme‐A reductase inhibitor, is used to reduce cholesterol levels. Accumulating evidence has revealed the immunomodulatory and anti‐inflammatory effects of simvastatin that prevent cardiovascular diseases. In addition, the beneficial effects of statins on fibrosis of various organs have been reported. However, the functional effect of statins on dermal fibrosis of keloids has not yet been explored. The objective of this study was to determine whether simvastatin could affect dermal fibrosis associated with keloids. We examined the effect of simvastatin on transforming growth factor (TGF)‐β1‐induced production of type I collagen, connective tissue growth factor (CTGF or CCN2), and α‐smooth muscle actin (α‐SMA). Keloid fibroblasts were cultured and exposed to different concentrations of simvastatin in the presence of TGF‐β1, and the effects of simvastatin on TGF‐β1‐induced collagen and CTGF production in keloid fibroblasts were determined. The type I collagen, CTGF, and α‐SMA expression levels and the Smad2 and Smad3 phosphorylation levels were assessed by Western blotting. The effect of simvastatin on cell viability was evaluated by assessing the colorimetric conversion of 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide. Simvastatin suppressed TGF‐β1‐induced type I collagen, CTGF, and α‐SMA production in a concentration‐dependent manner. The TGF‐β1‐induced Smad2 and Smad3 phosphorylation levels were abrogated by simvastatin pretreatment. The inhibition of type I collagen, CTGF, and α‐SMA expression by simvastatin was reversed by geranylgeranyl pyrophosphate, suggesting that the simvastatin‐induced cellular responses were due to inhibition of small GTPase Rho involvement. A RhoA activation assay showed that preincubation with simvastatin significantly blocked TGF‐β1‐induced RhoA activation. The Rho‐associated coiled kinase inhibitor Y27632 abrogated TGF‐β1‐induced production of type I collagen, CTGF, and α‐SMA. However, Y27632 had no significant effect on TGF‐β1‐induced phosphorylation of Smad2 and Smad3. In conclusion, the present study suggests that simvastatin is an effective inhibitor of TGF‐β1‐induced type I collagen, CTGF, and α‐SMA production in keloid fibroblasts.     

Effects of basic fibroblast growth factor,transforming growth factor-β1, insulin-like growth factor-1, and insulin on human osteoarthritic articular cartilage explants

This study evaluated the effects of basic fibroblast growth factor, transforming growth factor-β1, insulin-like growth factor-1, and insulin on the incorporation of thymidine and sulfate in human osteoarthritic articular cartilage. Tissue explants were obtained from 11 patients undergoing total knee arthroplasty and were categorized as nonfibrillated or fibrillated cartilage. The explants were cultured for 22 days, with changes of medium and growth factor every 72 hours, and labeled with [³H]thymidine and [³⁵S]sulfate. Growth factors were used in the following concentrations: basic fibroblast growth factor at 1, 10, and 100 ng/ml; transforming growth factor-β1 at 0.5, 5, and 50 ng/ml; insulin-like growth factor-1 at 0.15, 1.5, and 15 ng/ml; and insulin at 0.05, 0.5, and 5 μg/ml. Basic fibroblast growth factor decreased thymidine incorporation to 70% and sulfate incorporation to less than 20% that of the growth factor-free controls. Transforming growth factor-β1 had no significant effect on thymidine incorporation, whereas the concentrations studied inhibited sulfate incorporation to approximately 40% that of the controls. At the concentrations tested, insulin-like growth factor-1 had no significant effect on incorporation of either thymidine or sulfate. In contrast, insulin significantly stimulated the incorporation of both. Compared with growth factor-free controls, insulin maximally increased thymidine incorporation by a factor (± EM) of 2.36 ± 0.47 and 1.69 ± 0.19 in nonfibrillated and fibrillated explants, respectively; sulfate incorporation was maximally increased 1.60 ± 0.24 and 1.92 ± 0.29-fold for nonfibrillated and fibrillated explants, respectively. Of the factors tested, insulin demonstrated the greatest promise for promoting a synthetic response that may contribute to the regeneration of osteoarthritic cartilage.     

Effect of insulin-like growth factor 1 and basic fibroblast growth factor on DNA synthesis and collagen production in cultured anterior cruciate ligament cells

This study was undertaken to investigate the effects of insulin-like growth factor 1 (IGF-1) and basic fibroblast growth factor (bFGF) on the DNA and matrix synthesis of cells out-grown from the anterior cruciate ligament (ACL). Five batches of ACL cells from five 8-week-old Japanese white rabbits were isolated and maintained in culture until the fifth passage. We analyzed the effects of various concentrations of IGF-1 (1–1000 ng/ml) on [³H]-thymidine uptake in the cells at the first and fifth passages, and collagen content in the cell layer at the third passage, in the presence or absence of bFGF (10ng/ml). In the absence of bFGF, IGF-1 caused a significant increase in the synthesis of DNA and collagen in the ACL cells. IGF-1 and bFGF, in combination, synergistically increased the DNA synthesis of ACL cells, whereas such synergistic enhancement was not observed in their, collagen production. The amounts of [³H]-thymidine incorporated into the cells incubated with IGF-1 (500ng/ml) and bFGF (10ng/ml) combined were 1.3–1.5 times greater at first passage and 1.3–1.9 times greater at fifth passage than the sum of these with the growth factors used individually. Based on this in vitro finding, we consider it clinically relevant that IGF-1 and bFGF, when used together, have the capability to enhance the primary healing of ruptured ACL. Presented at the 11th Annual Meeting for Orthopaedic Research of the Japanese Orthopaedic Association, Kagoshima, Japan, October 17, 1996.     

Influence of transforming growth factor β1 and other growth factors on basic fibroblast growth factor level and proliferation of cultured human prostate-derived fibroblasts

Basic fibroblast growth factor (bFGF) has been identified in the human prostate. The level of bFGF has been reported to be elevated in benign prostatic hyperplasia (BPH), compared with normal prostate, suggesting that the growth factor may play a role in this disease of the prostate. Basic FGF is a mitogen for cultured human prostate-derived fibroblasts (PF). PF also synthesize bFGF, suggesting that growth regulation of these cells may be under autocrine control. The current study was undertaken to identify factors that affect PF proliferation and bFGF expression. Transforming growth factor β1 (TGF-β1) inhibited PF proliferation. The inhibition by TGF-β1 was partially overcome by bFGF but not by epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor type 1 (IGF-1), or insulin. Incubation of PF with TGF-β1 increased bFGF mRNA and immunoreactive bFGF levels in a dose- and time-dependent fashion. None of the other growth factor studies affected bFGF levels. PF were also found to express TGF-β1 mRNA, the level of which was increased two- to fivefold by TGF-β1. These observations suggest that PF proliferation is controlled by the interaction of two different growth factors. It is possible that bFGF/TGF-β imbalance in favor of cell proliferation promotes prostatic stromal hyperplasia. © 1993 Wiley-Liss, inc.     

Recombinant human platelet-derived growth factor and transforming growth factor-β mediated contraction of human dermal fibroblast populated lattices is inhibited by Rho/GTPase inhibitor but does not require phosphatidylinositol-3'' kinase

Matrix reorganization and tissue contraction are essential for wound healing. However, the intracellular signals that mediate these processes are largely unknown. We investigated cytokine-induced signaling and its potential role in contraction of adult human dermal fibroblast populated collagen lattices. The results document that recombinant human platelet-derived growth factor-BB and transforming growth factor-1 individually stimulate contraction of fibroblast populated collagen lattices, while a combination of the two cytokines leads to increased contraction. Although recombinant human platelet-derived growth factor-BB promoted collagen contraction, it failed to stimulate phosphatidylinositol-3' kinase in the human dermal fibroblasts. An inhibitor for phosphatidylinositol 3' kinase, wortmannin, had no effect on the cytokine-mediated collagen contraction. In addition, this failed activation of phosphatidylinositol 3' kinase is consistent with absence of tyrosine phosphorylation of the platelet-derived growth factor receptor when the cells are in a collagen matrix. In contrast, tyrosine phosphorylation of the platelet-derived growth factor receptor was readily detected in the dermal fibroblasts in monolayers. We also probed the potential role of Rho/GTPase in the cytokine-mediated contraction of fibroblast populated collagen lattices. Toxin B from Clostridium difficile at picomolar concentrations blocked both recombinant human platelet-derived growth factor and transforming growth factor-5 induced contraction. Further, this inhibition was correlated with the inhibition of cell spreading in collagen, which suggests the formation of actin fibers inside the cells is essential for cytokine-mediated contraction of fibroblast populated collagen lattices. Taken together, these results imply that Rho/GTPase signaling but not phosphoinositol-3' kinase is involved in the cytokine-mediated contraction of fibroblasts populated collagen lattice. These findings suggest a potential mechanism for these signaling components during human wound contraction.