Fibroblast Α-smooth Muscle Actin Research Articles

Motion lubrication suppressed mechanical activation via hydrated fibrous gene patch for tendon healing.

Mechanical activation of fibroblasts, caused by friction and transforming growth factor-β1 recognition, is one of the main causes of tissue adhesions. In this study, we developed a lubricated gene-hydrogel patch, which provides both a motion lubrication microenvironment and gene therapy. The patch's outer layer is composed of polyethylene glycol polyester hydrogel. The hydrogel forms hydrogen bonds with water molecules to create the motion lubrication layer, and it also serves as a gene delivery library for long-term gene silencing. Under the motion lubricated microenvironment, extracellular signal-regulated kinase-small interfering RNA can silence fibroblasts and enhance the blocking effect against fibroblast activation. In vitro, the proposed patch effectively inhibits fibroblast activation and reduces the coefficient of friction. In vivo, this patch reduces the expression of vimentin and α-smooth muscle actin in fibroblasts. Therefore, the lubricated gene-hydrogel patch can inhibit the mechanical activation of fibroblasts to promote tendon healing.

TIMP-1 Induces α-Smooth Muscle Actin in Fibroblasts to Promote Urethral Scar Formation

Background/Aims: Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been reported to upregulate in urethral scar. However, the underlying molecular mechanisms remain undefined. Methods: Here, we studied levels of TIMP-1 and α-smooth muscle actin (α-SMA) in the fibroblasts isolated from urethral scar tissues, compared to the fibroblasts isolated from normal urethra. Then we either overexpressed TIMP-1, or inhibited TIMP-1 by lentiviruses carrying a transgene or a short hairpin small interfering RNA for TIMP-1 in human fibroblasts. We examined the effects of modulation of TIMP-1 on α-SMA, and on epithelial-mesenchymal transition (EMT)-related genes. We also studied the underlying mechanisms. Results: We detected significantly higher levels of TIMP-1 and α-smooth muscle actin (α-SMA) in the fibroblasts isolated from urethral scar tissues, compared to the fibroblasts isolated from normal urethra. Moreover, the levels of TIMP-1 and α-SMA strongly correlated. Moreover, we found that TIMP-1 significantly increased levels of α-SMA, transforming growth factor β 1 (TGFβ1), Collagen I and some other key factors related to an enhanced EMT, suggesting that TIMP-1 may induce transformation of fibroblasts into myofibroblasts to promote tissue EMT to enhance the formation of urethral scar. Moreover, increases in TIMP-1 also induced an increase in fibroblast cell growth and cell invasion, in an ERK/MAPK-signaling-dependent manner. Conclusion: Our study thus highlights a pivotal role of TIMP-1 in urethral scar formation.

Thyrotropin-releasing hormone and its analogs accelerate wound healing

BackgroundThyrotropin-releasing hormone (TRH) is a classical hormone that controls thyroid hormone production in the anterior pituitary gland. However, recent evidence suggested that TRH is expressed in nonhypothalamic tissues such as epidermal keratinocytes and dermal fibroblasts, but its function is not clear. This study aimed to investigate the effects of TRH and its analogs on wound healing and explore the underlying mechanisms. Materials and methodsA stented excisional wound model was established, and the wound healing among vehicle control, TRH, and TRH analog taltirelin treatment groups was evaluated by macroscopic and histologic analyses. Primary fibroblasts were isolated from rat dermis and treated with vehicle control, TRH or taltirelin, cell migration, and proliferation were examined by scratch migration assay, MTT, and 5-ethynyl-2′- deoxyuridine (EdU) assay. The expression of α-Smooth muscle actin in fibroblasts was detected by Western blot and immunocytochemical analysis. ResultsTRH or taltirelin-treated wounds exhibited accelerated wound healing with enhanced granulation tissue formation and increased re-epithelialization and tissue formation. Furthermore, TRH or taltirelin promoted the migration and proliferation of fibroblasts and induced the expression of α-Smooth muscle actin in fibroblasts. ConclusionsTRH is important in upregulating the phenotypes of dermal fibroblasts and plays a role in accelerating wound healing.

The Compliance of Collagen Gels Regulates Transforming Growth Factor-β Induction of α-Smooth Muscle Actin in Fibroblasts

Wound contraction is mediated by myofibroblasts, specialized fibroblasts that appear in large numbers as the wound matures and when resistance to contractile forces increases. We considered that the regulation of myofibroblast differentiation by wound-healing cytokines may be dependent on the resistance of the connective tissue matrix to deformation. We examined transforming growth factor-beta1 (TGF-beta1) induction of the putative fibroblast contractile marker, alpha-smooth muscle actin (alpha-SMA), and the regulation of this process by the compliance of collagen substrates. Cells were cultured in three different types of collagen gels with wide variations of mechanical compliance as assessed by deformation testing. The resistance to collagen gel deformation determined the levels of intracellular tension as shown by staining for actin stress fibers. For cells plated on thin films of collagen-coated plastic (ie, minimal compliance and maximal intracellular tension), TGF-beta1 (10 ng/ml; 6 days) increased alpha-SMA protein content by ninefold as detected by Western blots but did not affect beta-actin content. Western blots of cells in anchored collagen gels (moderate compliance and tension) also showed a TGF-beta1-induced increase of alpha-SMA content, but the effect was greatly reduced compared with collagen-coated plastic (<3-fold increase). In floating collagen gels (high compliance and low tension), there were only minimal differences of alpha-SMA protein. Northern analyses for alpha-SMA and beta-actin indicated that TGF-beta1 selectively increased mRNA for alpha-SMA similar to the reported protein levels. In pulse-chase experiments, [35S]methionine-labeled intracellular alpha-SMA decayed most rapidly in floating gels, less rapidly in anchored gels, and not at all in collagen plates after TGF-beta1 treatment. TGF-beta1 increased alpha2 and beta1 integrin content by 50% in cells on collagen plates, but the increase was less marked on anchored gels and was undetectable in floating gels. When intracellular tension on collagen substrates was reduced by preincubating cells with blocking antibodies to the alpha2 and beta1 integrin subunits, TGF-beta1 failed to increase alpha-SMA protein content in all three types of collagen matrices. These data indicate that TGF-beta1-induced increases of alpha-SMA content are dependent on the resistance of the substrate to deformation and that the generation of intracellular tension is a central determinant of contractile cytoskeletal gene expression.

Myofibroblasts in the healing lapine medial collateral ligament: Possible mechanisms of contraction

The specific objective of this study was to determine the chronology of the appearance of the myofibroblast in the healing ligament. The overall goal of our work is to elucidate the cellular mechanism of contraction in this tissue. The myofibroblast has been found to be responsible for wound contraction in many tissues and to be the cause of the contracture in several pathological conditions. This cell type contains the actin isoform previously thought to be unique to smooth muscle cells and displays certain characteristic features at the ultrastructural level. In 26 New Zealand White male rabbits, the right medial collateral ligament was transected, whereas the left medial collateral ligament received a sham operation. The central third of the ligament (ligament scar tissue) was evaluated at 2, 3, 6, 8, 10, and 12 weeks postoperatively by immunohistochemical techniques, transmission electron microscopy, and Western blot analyses. Three other rabbits served as anatomic controls. During the early reparative phase (2 and 3 weeks after transection), there was an increase in the number of cells containing alpha-smooth muscle actin as well as augmentation of the alpha-smooth muscle actin content within each cell--a finding attributed to smooth muscle cells and pericytes associated with neovascularity. No myofibroblasts were detected at this stage, immediately postoperatively, or in the sham-operation controls. Ligaments in the remodeling phase of healing (6, 8, 10, and 12 weeks) exhibited alpha-smooth muscle actin in fibroblasts (myofibroblasts) as well as in vascular pericytes and smooth muscle cells. During this stage of healing, transmission electron microscopy demonstrated an increase in the number of cells displaying myofibroblastic features. It was estimated that at 12 weeks of healing 10% of the cells at the site of injury were myofibroblasts. This is the first definitive finding of myofibroblasts in the injury site of the healing ligament, to our knowledge. The appearance of myofibroblasts in the 6-12 week healing period, the interval during which the ligament has been shown to contract in studies by other investigators, is a rationale for a hypothesis that a cellular contractile apparatus comprising alpha-smooth muscle actin (i.e., the myofibroblast) may contribute to the recovery of original ligament length (and normal in situ strain).