Transforming-growth factor-beta (TGF-beta) is a pleiotrophic growth factor that is synthesized by many cells in the body. This growth factor is chemotactic for fibroblasts, stimulates fibroblast proliferation, and increases the synthesis of a number of extracellular matrix proteins including collagens. The TGF-beta activator protein is a transacting factor, which binds to the TGF-beta element in the distal promoter of the COL1A1 collagen gene and induces transcription of this gene. Although transient TGF-beta 1 activity participates in repair and regeneration of tissues, persistent TGF-beta 1 function affects excessive fibrosis and ultimately scarring of both skin and internal organs. Scarring of internal organ (e.g., liver and lung) results in a loss of function and ultimately death may occur. The central issue of this review is that phosphorothioate double-stranded decoys or other decoys decrease procollagen gene expression, procollagen synthesis, and collagen during fibrogenesis. The rationale is that the decoys containing the TGF-beta element or other gene transcription regulatory CIS-elements bind the transacting proteins preventing the latter from binding to the CIS-element in the 5'-flanking region of the natural gene resulting in transcription inhibition. We will, in part, focus on aspects involved in TGF-beta 1-induced fibrosis that occur during fibrogenesis and the use of the dsTGF-beta element containing oligodeoxynucleotide decoys to control excessive collagen synthesis, and deposition resulting from persistent TGF-beta. In our model of regulation of collagen synthesis, these double-stranded oligo decoys act as promoter competitors, binding to the activator protein either in the cytoplasm or in the nucleus. The significance of the proposed studies is that these novel natural antifibrotics will mimic the effect of glucocorticoids on collagen synthesis during fibrogenesis without the unwanted side effects of these steroids. Based on our previous studies on the molecular mechanisms by which glucocorticoids selectively decrease collagen synthesis, designed phosphorothioate oligodeoxynucleotides resistant to nuclease action will mimic the effects of glucocorticoids at the molecular, cellular, and in vivo levels of collagen synthesis. However, the glucocorticoids significantly inhibit noncollagen protein synthesis. Both the single-stranded and double-stranded oligodeoxynucleotide specifically decrease collagen synthesis without an inhibitory effect on noncollagen protein synthesis. In this review, we will specifically ask if TGF-beta-induced collagen synthesis is inhibited in cell culture and in vivo by using the double-stranded oligodeoxynucleotide decoys, will this inhibit fibrogenesis and ultimately scarring?
This review describes normal and abnormal wound healing, the latter characterized by excessive fibrosis and scarring, which for lung can result in morbidity and sometimes mortality. The cells, the extracellular matrix (ECM) proteins, and the growth factors regulating the synthesis, degradation, and deposition of the ECM proteins will be discussed. Therapeutics with particular emphasis given to gene therapies and their effects on specific signaling pathways are described. Bleomycin (BM), a potent antineoplastic antibiotic increases TGFb1 transcription, TGF-b1 gene expression, and TGF-b protein. Like TGF-b1, BM acts through the same distal promoter cis-element of the COL1A1 gene causing increased COL1 synthesis and lung fibrosis. Lung fibroblasts exist as subpopulations with one subset predominately responding to fibrogenic stimuli which could be a specific cell therapeutic target for the onset and development of pulmonary fibrosis.J TGF-b1 is an important modulator in the acute repair phase of a wound. It is a profibrotic factor that affects many cellular functions, including fibroblast proliferation and chemotaxis, stimulating the synthesis and deposition of connective tissue, and the inhibition of connective tissue breakdown. Type I collagen (COL 1) is the major fibrous collagen synthesized by wound fibroblasts in the repair process. When levels of TGF-b1 are chronically elevated, excessive fibrosis results which is characterized by increased collagen deposition. Fibrosis is the response to a traumatic event which terminates in the deposition of a connective tissue matrix. The composition and volume of that matrix differentiates between normal scar in acute wound repair and excessive fibrosis from chronic inflammation. In general, excess fibrosis contains increased concentrations of collagen, a rich blood supply, and myofibroblasts, identified by a-smooth muscle (SM) actin in cytoplasmic stress fibers. The development of fibrosis follows the sequence of overlapping phases; lag, proliferative, and remodeling. Either prolonging the proliferative phase and/or hindering the remodeling phase causes excess fibrosis. Trauma initiates the repair process that can terminate in a normal flat scar or a raised hypertrophic scar. The acute wound repair process provides a rapid and efficient way to restore mechanical tissue integrity. On the other hand, excess fibrosis is a prolonged process that can terminate into functional morbidity. An early event in repair is the restoration of homeostasis by the deposition of a fibrin clot which forms the highway for the migration of cells into the defect. At the termination of fibrosis, the fibrin matrix is replaced with a collagen matrix. The initial cells to travel the fibrin highway are the inflammatory cells. First, neutrophils enter the wound site followed by macrophages. Inflammatory cells prevent microbial colonization, remove dead tissue, and release factors that promote the proliferative phase. Fibroblasts and endothelial cells migrate into the wound site, where they synthesiz...
Transforming growth factor beta 1 (TGF-β1) plays a key role in connective tissue remodeling, scarring, and fibrosis. The effects of mechanical forces on TGF-β1 and collagen deposition are not well understood. We tested the hypothesis that brief (10 min) static tissue stretch attenuates TGF-β1-mediated new collagen deposition in response to injury. We used two different models: (1) an ex vivo model in which excised mouse subcutaneous tissue (N = 44 animals) was kept in organ culture for 4 days and either stretched (20% strain for 10 min 1 day after excision) or not stretched; culture media was assayed by ELISA for TGF-β1; (2) an in vivo model in which mice (N = 22 animals) underwent unilateral subcutaneous microsurgical injury on the back, then were randomized to stretch (20-30% strain for 10 min twice a day for 7 days) or no stretch; subcutaneous tissues of the back were immunohistochemically stained for Type-1 procollagen. In the ex vivo model, TGF-β1 protein was lower in stretched versus non-stretched tissue (repeated measures ANOVA, P < 0.01). In the in vivo model, microinjury resulted in a significant increase in Type-1 procollagen in the absence of stretch (P < 0.001), but not in the presence of stretch (P = 0.21). Thus, brief tissue stretch attenuated the increase in both soluble TGF-β1 (ex vivo) and Type-1 procollagen (in vivo) following tissue injury. These results have potential relevance to the mechanisms of treatments applying brief mechanical stretch to tissues (e.g., physical therapy, respiratory therapy, mechanical ventilation, massage, yoga, acupuncture).Transforming growth factor β1 (TGF-β1) is well-established as one of the key cytokines regulating the response of fibroblasts to injury, as well as the pathological production of fibrosis (Barnard et al., 1990;Sporn and Roberts, 1990;Leask and Abraham, 2004). Tissue injury is known to cause auto-induction of TGF-β1 protein production and secretion (Van Obberghen-Schilling et al., 1988;Morgan et al., 2000). Elevated extracellular levels of TGF-β1 have a major impact on extracellular matrix composition by causing autocrine and paracrine activation of fibroblast cell surface receptors, leading to increased synthesis of collagens, elastin, proteoglycans, fibronectin, and tenascin (Balza et al., 1988;Bassols and Massague, 1988;Kahari et al., 1992;Cutroneo, 2003). In vivo, connective tissue remodeling is not limited to tissue injury, but also occurs in response to changing levels of tissue mechanical forces (e.g., immobilization, beginning a new exercise or occupation). Longstanding physical therapy practices also suggest that externally applied mechanical forces can be used to reduce collagen deposition during tissue repair and scar formation (Cummings and Tillman, 1992). The mechanisms underlying these effects, however, are not well understood. In this study, we have used an ex vivo mouse subcutaneous tissue explant model and an in vivo mouse microinjury model to examine the effect of applying brief (10 min), static mechanical stretch on TGF-β...
In response to tissue injury connective tissue synthesis occurs either normally or abnormally, which is mediated by transforming growth factor-beta (TGF-beta) and other growth factors. This article will be primarily concerned with the response of injured tissues at the gene level of Type I procollagen synthesis in response to TGF-beta. This leads to provisional repair, which in turn may lead to involution, remodeling, regeneration, and ultimately repair. Alternately, continuation of provisional repair may lead to fibrosis and ultimately scarring. Scarring of internal organs such as the liver and the lung leads to loss of function and ultimately death. In the case of scarring of skin, this is a cosmetic problem and can be rectified by surgery. Type I procollagen is synthesized by two genes, proalpha1 (Type I) and proalpha2 (Type I) collagen genes. This article will focus on DNA binding sites on these two genes, which regulate the transcription of the specific gene. This article will also define specific cell signaling pathways for the turning on of the proalpha1 and proalpha2 (Type I) collagen genes. This article will address several questions. First, what is the major cytokine acting extracellularly which stimulates the transcription of the proalpha1 and proalpha2 (Type I) collagen genes during tissue fibrosis? Secondly, how are the signals transmitted by the extracellular profibrotic cytokine TGF-beta from the cellular membrane to the nucleus for transcription of the proalpha1 (Type I) and proalpha2 (Type I) collagen genes? Thirdly, what signaling pathways cross-talk with the signaling pathways resulting in the expression of the Type I collagen genes? Fourthly, how does TGF-beta affect extracellular matrix homeostasis? Fifthly, what are the nuclear factors corresponding to the DNA elements required for the promotion of the proalpha1 (Type I) and proalpha2 (Type I) collagen genes? Finally, how are the proalpha1 (Type I) and proalpha2 (Type I) collagen genes coordinately regulated? Strategies will also be presented for reducing fibrosis, which is the result of overexpression of TGF-beta.
Pulmonary fibrosis is a well-known toxic response to bleomycin treatment. Here we demonstrate the direct effects of bleomycin on lung fibroblasts that resulted in a marked increase of collagen synthesis as compared with total noncollagen protein synthesis. Bleomycin treatment of rat lung fibroblast cultures resulted in an increase of total cellular transforming growth factor-beta (TGF-beta) mRNA and increased secretion of TGF-beta protein into the conditioned media. beta 2-Microglobulin was measured as an mRNA that did not increase with bleomycin treatment. The bleomycin-induced increase of TGF-beta mRNA was decreased by cells cultured in the presence of either cycloheximide, an inhibitor of protein synthesis, or 2-mercapto-1-(beta-4-pyridethyl) benzimidazole, an inhibitor of RNA synthesis. To assess the mechanism underlying increased steady-state mRNA levels, the nuclear fraction was isolated from bleomycin-treated cells and the TGF-beta transcripts were determined. Transcription of TGF-beta mRNA was increased 12 h after bleomycin treatment, whereas the transcription of type I procollagen, type III procollagen, and beta-actin mRNAs were increased after 48 h of bleomycin treatment. beta 2-Microglobulin mRNA synthesis was not increased within this time frame. These results suggest bleomycin regulation of TGF-beta at both the mRNA and protein levels. Rats lung fibroblasts were separated by cell sorting into two subpopulations. One population of fibroblasts demonstrated increased procollagen type I mRNAs, whereas fibroblasts in the other population had increased procollagen type III mRNA. Following bleomycin treatment, TGF-beta mRNA was shown to be located more prominently in those fibroblasts that contain primarily collagen type I mRNAs.
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