transforming-growth-factor-beta and Contracture

Fibrillins constitute a family of large extracellular glycoproteins which multimerize to form microfibrils, an important structure in the extracellular matrix. It has long been assumed that fibrillin-2 was barely present during postnatal life, but it is now clear that fibrillin-2 molecules form the structural core of microfibrils, and are masked by an outer layer of fibrillin-1. Mutations in fibrillins give rise to heritable connective tissue disorders, including Marfan syndrome and congenital contractural arachnodactyly. Fibrillins also play an important role in matrix sequestering of members of the transforming growth factor-β family, and in context of Marfan syndrome excessive TGF-β activation has been observed. TGF-β activation is highly dependent on integrin binding, including integrin αvβ8 and αvβ6, which are upregulated upon TGF-β exposure. TGF-β is also involved in tumor progression, metastasis, epithelial-to-mesenchymal transition and tumor angiogenesis. In several highly vascularized types of cancer such as hepatocellular carcinoma, a positive correlation was found between increased TGF-β plasma concentrations and tumor vascularity. Interestingly, fibrillin-1 has a higher affinity to TGF-β and, therefore, has a higher capacity to sequester TGF-β compared to fibrillin-2. The previously reported downregulation of fibrillin-1 in tumor endothelium affects the fibrillin-1/fibrillin-2 ratio in the microfibrils, exposing the normally hidden fibrillin-2. We postulate that fibrillin-2 exposure in the tumor endothelium directly stimulates tumor angiogenesis by influencing TGF-β sequestering by microfibrils, leading to a locally higher active TGF-β concentration in the tumor microenvironment. From a therapeutic perspective, fibrillin-2 might serve as a potential target for future anti-cancer therapies.

Topics: Animals; Arachnodactyly; Connective Tissue; Contracture; Disease Models, Animal; Endothelium, Vascular; Fibrillin-2; Humans; Marfan Syndrome; Mutation; Neoplasms; Neovascularization, Pathologic; Transforming Growth Factor beta; Tumor Microenvironment

Dysregulated wound healing and pathologic fibrosis cause abnormal scarring, leading to poor functional and aesthetic results in hand burns. Understanding the underlying biologic mechanisms involved allows the hand surgeon to better address these issues, and suggests new avenues of research to improve patient outcomes. In this article, the authors review the biology of scar and contracture by focusing on potential causes of abnormal wound healing, including depth of injury, cytokines, cells, the immune system, and extracellular matrix, and explore therapeutic measures designed to target the various biologic causes of poor scar.

Topics: Animals; Cicatrix; Contracture; Cytokines; Fibroblasts; Humans; Keratinocytes; Platelet-Derived Growth Factor; Splints; T-Lymphocytes; Transforming Growth Factor beta; Wound Healing

Although a substantial amount of molecular and cellular data have been generated in an effort to understand the process of wound contraction and scar contracture formation, questions remain. What seems apparent is that the myofibroblast is not the only cell that generates contractile forces within wounds, but it does appear to be intrinsically linked to the development of hypertrophic scars. The supposition that the formation of scar contractures is solely the result of a continuation of wound contraction is an oversimplification. Figure 4 provides a model of the possible evolution of contractile forces during the wound healing process and their role in the development of scar contractures. Migration of fibroblasts into and through the extracellular matrix during the initial phase of wound healing, prior to the expression of alpha-SMA, appears to be a fundamental component of wound contraction. During this migration, the pulling of collagen fibrils into a streamlined pattern in their wake, and the associated production of collagenase, may facilitate a more normal arrangement of collagen. Once the wound has been repopulated and the chemotactic gradient that was established by inflammatory cells is decreased, fibroblast migration will cease. It is at this point that myofibroblasts appear and play a key role in the production of hypertrophic scars, given that their prolonged presence and over-representation are hallmarks of this pathology. One of the pivotal differences between wounds that proceed to normal scar compared with those that develop hypertrophic scars and scar contractures may be a lack (or late induction) of myofibroblast apoptotic cell death. The combined contribution of fibroblasts and myofibroblasts to abnormal extracellular matrix protein production results in an excessive and rigid scar. The isometric application of contractile forces by myofibroblasts probably contributes to the formation of the whorls, nodules, and scar contractures characteristic of hypertrophic scars. Because the prolonged presence of myofibroblasts, producing an imbalance in extracellular matrix proteins and proteases, probably exacerbates hypertrophic scars and wound contraction, accelerating the rate of apoptotic cell death to reduce the cell number to that seen in normal scar may be a useful strategy for providing effective and efficient treatment of scar contracture.

Topics: Cicatrix, Hypertrophic; Collagen; Contracture; Extracellular Matrix; Fibroblasts; Humans; Skin; Transforming Growth Factor beta; Wound Healing

Bladder neck contracture (BNC), one of the most challenging complications after transurethral resection of the prostate (TURP) and photoselective vaporization of the prostate (PVP), lacks effective treatment. In the present study, our experience in treating BNC using GreenLight laser vaporization with triamcinolone acetonide (TA) injections was shared. This is a retrospective cohort study that included 46 patients with BNC after TURP and PVP in our center. GreenLight laser surgeries (180 W) were carried out and TA was administrated simultaneously. TA injections were repeated every week for three times after surgeries. The perioperative and postoperative parameters were reviewed and compared. Bladder neck tissues were examined by immunohistochemical staining to explore the expressions of collagen I, matrix metalloproteinase-3 (MMP-3), and transforming growth factor-β (TGF-β) after treatments. The chief complaint symptoms of all patients were significantly relieved after our treatments. None of them showed BNC recurrence during the follow-up. Complications were rare and mild. Postoperative assessments including maximal urinary flow rate (P < 0.01), International Prostate Symptom Score (P < 0.01), quality of life index (P < 0.01), and post-void residual volume (P < 0.001) were significantly better than baseline values, respectively. Immunohistochemical staining showed significantly lower expressions of collagen I (P < 0.001), MMP-3 (P < 0.001), and TGF-β (P < 0.001) after treatments. In conclusion, 180-W GreenLight laser with repeated TA injections demonstrated the safety and long-term efficacy in treating BNC, by inhibiting the expressions of fibrotic factors. Our procedure was a promising treatment for BNC after PVP and TURP.

Topics: Collagen; Contracture; Humans; Laser Therapy; Lasers; Male; Matrix Metalloproteinase 3; Prostatic Hyperplasia; Quality of Life; Retrospective Studies; Transforming Growth Factor beta; Transforming Growth Factors; Transurethral Resection of Prostate; Treatment Outcome; Triamcinolone Acetonide; Urinary Bladder; Volatilization

To explore the role of transforming growth factor-β (TGF-β) in bladder neck contracture (BNC) after transurethral enucleation and resection of the prostate (TUERP).. This study included 300 BPH patients undergoing TUERP, aged 51－89 (69.19 ± 8.43) years, with the prostate volume of 14.4－355.8 (63.18 ± 47.63) ml and preoperative IPSS of 15－35 (26.07 ± 5.9), QOL score of 3－6 (4.43 ± 0.67), PSA content of 0.17－23.16 (2.94 ± 3.77) ug/L, urinary leukocyte increase in 50 cases, post-void residual urine volume (PVR) of 0－440 (83.53 ± 86.85) ml, and maximum urinary flow rate (Qmax) of 2.3－14.5 (7.77 ± 3.47) ml/s. During TUERP, we collected the tissues from the bladder neck at 5 and 7 o'clock as well as the BPH tissue and the tissue from the residual prostate for HE staining, immunohistochemistry (the SP method) and examination of the infiltration degree of inflammatory cells and expressions of TGF-β1 and TGF-β3. During the 6－24 months follow-up, 6 of the patients were confirmed with BNC based on the clinical symptoms and the results of uroflowmetry and cystoscopy, and underwent transurethral bladder neck incision and detection of the expressions of TGF-β1 and TGF-β3 in the bladder neck tissue with BNC.. The bladder neck tissue without BNC was mainly composed of smooth muscle and fibrous tissues with local infiltration of inflammatory cells, and the residual prostate tissue primarily comprised fibrous and muscle tissues, mixed with a little prostatic epithelial tissue. The bladder neck tissue with BNC, compared with that harvested during the initial TUERP, exhibited significantly increased expression of TGF-β1 (［68.20 ± 10.88］% vs ［36.14 ± 7.62］%, P < 0.05), decreased expression of TGF-β3 (［8.55 ± 4.73］% vs ［20.77 ± 8.69］%, P < 0.05), and enhanced infiltration of inflammatory cells (P < 0.05). The bladder neck tissue without BNC, in comparison with the BPH tissue, showed dramatically up-regulated expressions of TGF-β1 (［27.05 ± 8.21］% vs ［1.61 ± 0.69］%, P < 0.001) and TGF-β3 (［14.09 ± 4.19］% vs ［0.32 ± 0.11］%, P < 0.001) and increased infiltration of inflammatory cells (P < 0.05).. After TUERP, the expression of TGF-β1 is increased, that of TGF-β3 decreased and the infiltration of inflammatory cells enhanced in the bladder neck tissue with BNC, which suggests that BNC may be related to the expression of TGF-β and that BNC after TUERP could be prevented by regulating the expression of TGF-β.

Topics: Aged; Aged, 80 and over; Contracture; Humans; Male; Middle Aged; Prostate; Quality of Life; Transforming Growth Factor beta; Urinary Bladder

Joint contracture is a common complication of joint injury. This study aimed to assess the effect of inhibiting the transforming growth factor-β (TGF-β) signaling during joint immobilization and remobilization on immobilization-induced joint contracture in rats.. The knees of rats were immobilized using Kirschner wires following trauma to the femoral condyles to generate joint contracture. After immobilization, levels of TGF-β and passive extension range of motion (ROM) were measured at different time points, joints were histologically analyzed by hematoxylin and eosin (H&E) and Masson trichrome staining, and the expression of inflammatory or fibrosis-related mediators, including interleukin-1β (IL-1β), phosphorylated Smad2/3 (p-Smad2/3), α-smooth muscle actin (α-SMA) and collagen types I (Col 1) and III (Col 3), were examined in joint capsules using immunohistochemistry and quantitative real-time polymerase chain reaction (qRT-PCR). Rats were also treated with LY2157299, a TGF-β receptor I kinase inhibitor, at different stages of immobilization and remobilization.. TGF-β1 levels in the serum and the number of p-Smad2/3. Inhibiting TGF-β signaling paired with active motion effectively attenuated the formation of immobilization-induced joint contracture in rats.

Topics: Animals; Contracture; Joint Capsule; Knee Joint; Range of Motion, Articular; Rats; Transforming Growth Factor beta; Transforming Growth Factor beta1

Joint contractures are a major complication in patients with spinal cord injuries. Positioning, stretching, and physical therapy are advocated to prevent and treat contractures; however, many patients still develop them. Joint motion (exercise) is crucial to correct contractures. Transcutaneous carbon dioxide (CO2) therapy was developed recently, and its effect is similar to that of exercise. This therapy may be an alternative or complementary approach to exercise.. Using an established model of spinal cord injury in rats with knee flexion contractures, we sought to clarify whether transcutaneous CO2 altered (1) contracture, as measured by ROM; (2) muscular and articular factors contributing to the loss of ROM; (3) fibrosis and fibrosis-related gene expression in muscle; and (4) the morphology of and fibrosis-related protein expression in the joint capsule.. Thirty-six Wistar rats were divided into three equal groups: caged control, those untreated after spinal cord injury, and those treated with CO2 after spinal cord injury. The rats were treated with CO2 from either the first day (prevention) or 15th day (treatment) after spinal cord injury for 2 or 4 weeks. The hindlimbs of rats in the treated group were exposed to CO2 gas for 20 minutes once daily. Knee extension ROM was measured with a goniometer and was measured again after myotomy. We calculated the muscular and articular factors responsible for contractures by subtracting the post-myotomy ROM from that before myotomy. We also quantified histologic muscle fibrosis and evaluated fibrosis-related genes (collagen Type 1, α1 and transforming growth factor beta) in the biceps femoris muscle with real-time polymerase chain reaction. The synovial intima's length was measured, and the distribution of fibrosis-related proteins (Type I collagen and transforming growth factor beta) in the joint capsule was observed with immunohistochemistry. Knee flexion contractures developed in rats after spinal cord injuries at all timepoints.. CO2 therapy improved limited-extension ROM in the prevention group at 2 weeks (22° ± 2°) and 4 weeks (29° ± 1°) and in the treatment group at 2 weeks (31° ± 1°) compared with untreated rats after spinal cord injuries (35° ± 2°, mean difference, 13°; 39° ± 1°, mean difference, 9°; and 38° ± 1°, mean difference, 7°, respectively) (95% CI, 10.50-14.86, 8.10-10.19, and 4.73-9.01, respectively; all p < 0.001). Muscular factors decreased in treated rats in the prevention group at 2 weeks (8° ± 2°) and 4 weeks (14°± 1°) and in the treatment group at 2 weeks (14 ± 1°) compared with untreated rats (15° ± 1°, 4.85-9.42; 16° ± 1°, 1.24-3.86; and 17° ± 2°, 1.16-5.34, respectively; all p < 0.05). The therapy improved articular factors in the prevention group at 2 weeks (4° ± 1°) and 4 weeks (6° ± 1°) and in the treatment group at 2 weeks (8° ± 1°) compared with untreated rats (10° ± 1°, 4.05-7.05; 12° ± 1°, 5.18-8.02; and 11° ± 2°, 1.73-5.50, respectively; all p < 0.05). CO2 therapy decreased muscle fibrosis in the prevention group at 2 weeks (p < 0.001). The expression of collagen Type 1, α1 mRNA in the biceps femoris decreased in treated rats in the prevention group at 2 and 4 weeks compared with untreated rat (p = 0.002 and p = 0.008, respectively), although there was little difference in the expression of transforming growth factor beta (p > 0.05). CO2 therapy did not improve shortening of the synovial intima at all timepoints (all p > 0.05). CO2 therapy decreased transforming growth factor beta immunolabeling in joint capsules in the rats in the prevention group at 2 weeks. The staining intensity and Type I collagen pattern showed no differences among all groups at all timepoints.. CO2 therapy may be useful for preventing and treating contractures after spinal cord injuries. CO2 therapy particularly appears to be more effective as a prevention and treatment strategy in early-stage contractures before irreversible degeneration occurs, as shown in a rat model.. Our findings support the idea that CO2 therapy may be able to improve the loss of ROM after spinal cord injury.

Topics: Administration, Cutaneous; Animals; Biomechanical Phenomena; Carbon Dioxide; Collagen Type I; Collagen Type I, alpha 1 Chain; Contracture; Disease Models, Animal; Fibrosis; Joints; Male; Muscle, Skeletal; Range of Motion, Articular; Rats, Wistar; Recovery of Function; Spinal Cord Injuries; Time Factors; Transforming Growth Factor beta

Gluteal muscle contracture (GMC) is a chronic fibrotic disease of gluteal muscles which is characterized by excessive deposition of collagen in the extracellular matrix. Transforming growth factor (TGF)-βs have been shown to play an important role in the progression of GMC. However, the underlying mechanisms are not entirely clear. We sought to explore the expression of TGF-β/Smad pathway proteins and their downstream targets in gluteal muscle contracture disease.. The expression levels of collagens type I/III, TGF-β1, Smad2/3/4/7 and PAI-1 (plasminogen activator inhibitor type 1) in gluteal muscle contraction (GMC) patients were measured using immunohistochemistry, reverse transcription and polymerase chain reaction (RT-PCR) and western blot assays.. The expressions of collagens type I/III and TGF-β1 were significantly increased in the contraction band compared with unaffected muscle. In addition, R-Smad phosphorylation and Smad4 protein expression in the contraction band were also elevated, while the expression of Smad7 was significantly decreased in the fibrotic muscle of the GMC patients compared to the unaffected adjacent muscle. The protein and mRNA levels of PAI-1 were also remarkably increased in the contraction band compared with adjacent muscle. Immunohistochemical analysis also demonstrated that the expression levels of TGF-β1 and PAI-1 were higher in contraction band than those in the adjacent muscle.. Our data confirm the stimulating effects of the TGF-β/Smad pathway in gluteal muscle contracture disease and reveal the internal changes of TGF-β/Smad pathway proteins and their corresponding targets in gluteal muscle contracture patients.

Topics: Adolescent; Adult; Buttocks; Collagen Type I; Collagen Type III; Contracture; Female; Humans; Immunohistochemistry; Male; Muscle, Skeletal; Phosphorylation; Plasminogen Activator Inhibitor 1; RNA, Messenger; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Up-Regulation; Young Adult

In systemic sclerosis (SSc), a common and aetiologically mysterious form of scleroderma (defined as pathological fibrosis of the skin), previously healthy adults acquire fibrosis of the skin and viscera in association with autoantibodies. Familial recurrence is extremely rare and causal genes have not been identified. Although the onset of fibrosis in SSc typically correlates with the production of autoantibodies, whether they contribute to disease pathogenesis or simply serve as a marker of disease remains controversial and the mechanism for their induction is largely unknown. The study of SSc is hindered by a lack of animal models that recapitulate the aetiology of this complex disease. To gain a foothold in the pathogenesis of pathological skin fibrosis, we studied stiff skin syndrome (SSS), a rare but tractable Mendelian disorder leading to childhood onset of diffuse skin fibrosis with autosomal dominant inheritance and complete penetrance. We showed previously that SSS is caused by heterozygous missense mutations in the gene (FBN1) encoding fibrillin-1, the main constituent of extracellular microfibrils. SSS mutations all localize to the only domain in fibrillin-1 that harbours an Arg-Gly-Asp (RGD) motif needed to mediate cell-matrix interactions by binding to cell-surface integrins. Here we show that mouse lines harbouring analogous amino acid substitutions in fibrillin-1 recapitulate aggressive skin fibrosis that is prevented by integrin-modulating therapies and reversed by antagonism of the pro-fibrotic cytokine transforming growth factor β (TGF-β). Mutant mice show skin infiltration of pro-inflammatory immune cells including plasmacytoid dendritic cells, T helper cells and plasma cells, and also autoantibody production; these findings are normalized by integrin-modulating therapies or TGF-β antagonism. These results show that alterations in cell-matrix interactions are sufficient to initiate and sustain inflammatory and pro-fibrotic programmes and highlight new therapeutic strategies.

Topics: Amino Acid Motifs; Amino Acid Substitution; Animals; Antibodies, Antinuclear; Antibodies, Neutralizing; Autoimmunity; Contracture; Dendritic Cells; Female; Fibrillin-1; Fibrillins; Fibrosis; Integrins; Male; Mice; Microfilament Proteins; Mutation, Missense; Plasma Cells; Scleroderma, Systemic; Skin Diseases, Genetic; T-Lymphocytes, Helper-Inducer; Transforming Growth Factor beta

Gluteal muscle contracture (GMC) is a multi-factor human chronic fibrotic disease of the gluteal muscle. Fibrotic tissue is characterized by excessive accumulation of collagen in the muscle's extracellular matrix. Transforming growth factor (TGF)-beta1 and -beta2 are thought to play an important role in fibrogenesis, while TGF-beta3 is believed to have an anti-fibrotic function. We hypothesize that the expression of collagen and TGF-betas would be up-regulated in GMC patients.. The expression of collagen type I, type III and TGF-betas were studied in 23 fibrotic samples and 23 normal/control samples in GMC patients using immunohistochemistry, reverse transcription and polymerase chain reaction (RT-PCR) and western bolt analysis.. Compared to the unaffected adjacent muscle, increased expression of TGF-beta1 and -beta3 was associated with deposition of collagen type I and type III in the fibrotic muscle of the GMC patients at the mRNA level. Strong up-regulation of these proteins in fibrotic muscle was confirmed by immunohistochemical staining and western blot analysis. TGF-beta2 was not up-regulated in relation to GMC.. This study confirmed our hypothesis that collagen types I, III, TGF-beta1 and TGF-beta3 were up-regulated in biopsy specimens obtained from patients with GMC. Complex interaction of TGF-beta1 with profibrotic function and TGF-beta3 with antifibrotic function may increase synthesis of collagens and thereby significantly contribute to the process of gluteal muscle scarring in patients with GMC.

Topics: Adolescent; Adult; Biomarkers; Buttocks; Child; Collagen; Collagen Type I; Collagen Type III; Contracture; Female; Fibrosis; Genetic Predisposition to Disease; Humans; Male; Muscle, Skeletal; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta3; Up-Regulation; Young Adult

Few studies emphasize the collagen metabolism-related cytokines and ultrastructure of the completely stress-shielded Achilles tendon. In this study, we used a rat model with complete stress shielding of the Achilles tendon to observe the changes in the ultrastructure of the Achilles tendon and concentration of IL-1 and TGF-β 3 weeks after stress shielding. The model group comprised 12 male Sprague-Dawley rats. The stress of the Achilles tendon of the left hind limb was shielded through tendon cerclage combined with sciatic nerve transection, and the right served as a normal control. Three weeks later, the ultrastructure of the Achilles tendon was observed under electron microscopy and IL-1 and TGF-β levels were determined by enzyme-linked immunosorbent assay. Compared with the control side, collagen fibrils of the shielded Achilles tendons were irregularly arranged and loose. The number of small-diameter collagen fibrils increased significantly with the decrease of the average diameter of collagen fibrils. At the same time, IL-1 concentrations increased significantly in the model group as compared to that in the control group, but no significant difference was found in TGF-β levels. These results suggest that IL-1 may play an important role in the change of ultrastructure after stress shielding.

Topics: Achilles Tendon; Animals; Collagen; Contracture; Disease Models, Animal; Fracture Fixation, Internal; Hindlimb; Interleukin-1; Male; Microscopy, Electron, Transmission; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Stress, Mechanical; Suture Techniques; Sutures; Tensile Strength; Transforming Growth Factor beta

Fibrosis of normal tissues often accompanies radiation treatment of cancer. Activation of the transforming growth factor-beta (TGF-beta) signaling pathway is thought to play a major role in radiation-induced fibrosis and has prompted the development and assessment of low molecular weight inhibitors of the pathway. Previous studies with halofuginone have shown it to inhibit TGF-beta signaling in vitro and protect mice from radiation-induced leg contraction (a model for soft tissue fibrosis). The current study confirms these findings for HaCaT cells stimulated with exogenous TGF-beta treatment. Reducing the halifuginone treatment from 7 days/week (used previously) to 5 days/week post-radiation exposure provided significant protection against radiation-induced leg contraction in mice 3 and 4 months post-radiation treatment. Halofuginone treatment was shown to attenuate TGF-beta signaling molecules taken from irradiated skin including TGF-betaRII, pSmad3, Smad7, and TSP1. The latter, TSP1, a co-activator of TGF-beta may serve as a suitable biomarker for monitoring the efficacy of halofuginone should it be evaluated in a clinical setting for protection against radiation-induced fibrosis.

Topics: Animals; Cell Line; Contracture; Female; Fibrosis; Humans; Leg; Mice; Mice, Inbred C3H; Piperidines; Protein Serine-Threonine Kinases; Quinazolinones; Radiotherapy; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta is a potent inducer of both transdifferentiation and contraction, which are regarded as critical processes that underpin tissue fibrosis. Consequently, transdifferentiation is believed to drive TGFbeta-mediated contraction. This study was conducted to determine the relationship between transdifferentiation of human lens epithelial cells and matrix contraction.. Real-time PCR was used to investigate gene expression of transdifferentiation markers in the human lens cell line FHL 124 and native lens epithelia. Contraction was assessed with a patch-contraction assay, whereby all areas covered by cells were measured with imaging techniques after fixation and cell staining with Coomassie blue. In addition, total protein content, determined by dye extractions was used to give an estimate of total cell population. To prevent fibronectin-fibronectin receptor interaction 100 microM RGDS peptide was used. Suppression of TGFbeta-induced alphaSMA expression was mediated by siRNA technology.. Real-time PCR analysis showed 10 ng/mL TGF-beta1 or -beta2 significantly increased expression of alphaSMA, fibronectin, and alpha5beta1 integrin (fibronectin receptor components) in FHL 124 cells and human lens epithelia. Cultures maintained in TGFbeta and RGDS showed a marked increase in the rate of contraction relative to TGF-beta alone. RGDS alone did not differ significantly from the control. Real-time PCR and Western blots showed reduced levels of message and alphaSMA protein when transfected with siRNA. alphaSMA knockdown did not prevent TGFbeta-induced contraction.. A targeted inhibition approach demonstrated that key elements associated with transdifferentiation are not critical for TGFbeta-induced matrix contraction.

Topics: Actins; Blotting, Western; Cell Line; Cell Transdifferentiation; Contracture; Dose-Response Relationship, Drug; Epithelial Cells; Fibronectins; Gene Expression; Humans; Integrin alpha5beta1; Lens, Crystalline; Oligopeptides; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Signal Transduction; Transfection; Transforming Growth Factor beta

To assess the significance of Rho-kinase-dependent contractility in TGF-beta-induced myofibroblast transdifferentiation of human tenon fibroblasts to characterize possible pharmacological targets for the inhibition of postoperative scarring after glaucoma surgery.. Human tenon fibroblasts (HTFs) were grown in culture and stimulated with TGF-beta1. The effect of TGF-beta on Rho-GTPase activity was assessed by GST-rhotekin binding domain pulldown assay and detected by Western blot analysis. Contractility was evaluated in a silicone substrate wrinkling assay and in fibroblast-populated collagen gels. The actin cytoskeleton and focal adhesions were visualized by immunofluorescence microscopy. alpha-SMA transcripts were measured by real-time RT-PCR. TGF-beta-induced Smad- and p38-activation and expression of alpha-SMA were detected by Western blot analysis. Nuclear translocation of Smad2/3 was determined by confocal immunofluorescence microscopy. The influence of Rho-dependent kinase (ROCK) and myosin light chain kinase (MLCK) were studied by using specific kinase inhibitors (Y-27632, HA-1077, H-1152, and ML-7).. Within 10 minutes of stimulation, TGF-beta induced Rho activation that was associated with an increase in cell tension and followed by actin stress fiber enhancement. ROCK inhibitors released cell tension and averted TGF-beta-induced cytoskeletal changes, p38 activation and subsequent alpha-SMA expression, whereas Smad2-phosphorylation and nuclear translocation were preserved. MLCK inhibition also blocked alpha-SMA expression. In fibroblast-populated collagen lattices, ROCK inhibitors prevented TGF-beta-induced stress fiber assembly and contraction.. TGF-beta induces a rapid contractile response in HTFs that precedes myofibroblast transdifferentiation. ROCK inhibitors release this contraction and block subsequent TGF-beta-induced myofibroblast transdifferentiation and may therefore serve to modulate postoperative scarring after glaucoma filtering surgery.

Topics: Actins; Blotting, Western; Cell Differentiation; Cells, Cultured; Connective Tissue Cells; Contracture; Enzyme Inhibitors; Fibroblasts; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation; Humans; Intracellular Signaling Peptides and Proteins; Microscopy, Confocal; Myosin-Light-Chain Kinase; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Reverse Transcriptase Polymerase Chain Reaction; rho-Associated Kinases; RNA, Messenger; Smad2 Protein; Transforming Growth Factor beta