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Background/aims: Hypertrophic scars (HS) formation results from reduced apoptosis and increased proliferation of fibroblasts. Therefore, apoptosis of fibroblasts is a key target for the development of novel therapeutic strategies for HS. Previous reports demonstrated that FK506 could attenuate scar formation in vivo and FK506 could also induce endoplasmic reticulum stress (ER stress). However, the effects of FK506 on ER stress-mediated apoptosis in fibroblasts remain unclear., Methods: Rat skin fibroblasts were used in the study. Cell viability was examined using cell counting Kit-8. Apoptosis was detected by Annexin V/Propidium Iodide Double Staining. Gene silencing was performed using Small Interfering RNAs (siRNAs) or via lentiviral infection. The expression of apoptosis-related proteins was determined via Western blot. Interaction between proteins was explored by co-immunoprecipitation., Results: FK506 significantly reduced cell viability and induced apoptosis in fibroblasts. Interestingly, ER stress was also activated after FK506 treatment. We further demonstrated that FK506-induced apoptosis was mediated by ER stress via activating CHOP, evidenced by decreased apoptosis after inhibition of ER stress using TUDCA or silencing expression of CHOP. Furthermore, Co-immunoprecipitation results indicated that treatment of FK506 induced disassociation of FKBP12.6 from RyR2 and its translocation from ER membrane to cytosol, consequently promoting ER stress-mediated apoptosis., Conclusion: FK506-induced fibroblasts apoptosis was mediated by ER stress via CHOP signaling pathway., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Background/aims: Epidural fibrosis, a common complication after laminectomy, has been demonstrated to be closely associated with poor surgical outcomes. Previous studies showed that taurine had remarkable anti-fibrotic effects on lung and liver fibrosis. We performed this study to investigate the effects of taurine in rat models of epidural fibrosis after laminectomy and to explore the potential molecular mechanism., Methods: Laminectomy was performed on each rat to establish epidural fibrosis model. After taurine treatment, Masson's trichrome and immunohistochemistry staining were used to examine epidural fibrosis. Cell viability was determined using the Cell Counting Kit-8 assay. Annexin V/Propidium Iodide double staining was performed to detect fibroblasts apoptosis. Microarray was adopted to identify significantly changed mRNAs. mRNA expression was measured by qRT-PCR. Lentivirus infection was performed to establish stable knockdown and overexpression cell lines. The expression of fibrosis-related proteins was determined via Western blot., Results: Taurine treatment markedly reduced laminectomy-induced epidural fibrosis in rat models. However, this effect of taurine was independent on TGF-β/Smad pathway, evidenced by no change in the expression of TGF-β and its receptors. Besides, taurine had almost no effect on cell apoptosis. Interestingly, taurine treatment significantly decreased expression of EGR1 (Early growth response protein 1), an enhancer of fibrosis, both in vivo and in vitro. Furthermore, overexpression of EGR1 increased activation of fibroblasts, while EGR1 knockdown achieved an opposite effect, indicating that EGR1 plays a key role in the inhibitory effect of taurine on TGF-β-induced fibrosis., Conclusions: Reduced epidural fibrosis in vivo and decreased activation of fibroblasts in vitro after taurine treatment was mediated by EGR1. Taurine promises to be a potential prevention for epidural fibrosis after laminectomy., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Mitomycin C (MMC) is known to reduce epidural fibrosis, but the underlying mechanisms have not yet been elucidated. Aberrant miR-200b expressions have been reported in multiple types of fibrotic tissues from many diseases. The aim of this study was to clarify the mechanism by which MMC induces fibroblasts apoptosis and reduces epidural fibrosis. The expression of miR-200b in human fibroblasts was determined after MMC treatment, and the targeted association between miR-200b and RhoE was determined using the luciferase activity assay. The effects of MMC and miR-200b on human fibroblasts apoptosis were evaluated using flow cytometry and western blot analysis. The effects of MMC and miR-200b on epidural fibrosis were evaluated using the Rydell classification, hydroxyproline content, apoptotic cell count and histological analysis. The study revealed that MMC could significantly downregulate miR-200b expression and induce human fibroblasts apoptosis. The direct downregulation of miR-200b could induce human fibroblasts apoptosis. Furthermore, we identified the binding sequence for miR-200b within the 3' untranslated region of RhoE. RhoE was confirmed to be a direct target of miR-200b, and RhoE itself acted as a promoter of fibroblasts apoptosis. The inhibition of miR-200b increased fibroblasts apoptosis and reduced epidural fibrosis in rats, which was in accordance with the effect of MMC. This study suggests that MMC induces fibroblasts apoptosis and reduces epidural fibrosis by regulating miR-200b expression and its targeting of RhoE., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Endoplasmic reticulum (ER) stress-mediated cell apoptosis has been implicated in various cell types, including fibroblasts. Previous studies have shown that mitomycin C (MMC)-induced apoptosis occurs in fibroblasts, but the effects of MMC on ER stress-mediated apoptosis in fibroblasts have not been examined. Here, MMC-induced apoptosis in human primary fibroblasts was investigated by exposing cells to a single dose of MMC for 5 minutes. Significant inhibition of cell proliferation and increased apoptosis were observed using a cell viability assay, Annexin V/propidium iodide double staining, cell cycle analysis, and TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining. Upregulation of proapoptotic factors, including cleaved caspase-3 and poly ADP-ribose polymerase (PARP), was detected by Western blotting. MMC-induced apoptosis was correlated with elevation of 78-kDa glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP), which are hallmarks of ER stress. Three unfolded protein response (UPR) sensors (inositol-requiring enzyme 1, IRE1; activating transcription factor 6, ATF6; and PKR-like ER kinase, PERK) and their downstream signaling pathways were also activated. Knockdown of CHOP attenuated MMC-induced apoptosis by increasing the ratio of BCL-2/BAX and decreasing BIM expression, suggesting that ER stress is involved in MMC-induced fibroblast apoptosis. Interestingly, knockdown of PERK significantly decreased ER stress-mediated apoptosis by reducing the expression of CHOP, BIM and cleaved caspase-3. Reactive oxygen species (ROS) scavenging also decreased the expression of GRP78, phospho-PERK, CHOP, and BIM. These results demonstrate that MMC-induced apoptosis is triggered by ROS generation and PERK activation.

Background Context: Recovery from spine surgery is usually accompanied with the development of epidural scar adhesions from the abnormal proliferation of fibroblast and extracellular matrix-related metabolism. Polyethylene glycol (PEG) could alleviate the postsurgical adhesion formation with still leaving a gap between the sheet and the dura. However, PEG film could not function as a three-dimensional barrier to prevent adhesion completely. Mitomycin C (MMC) could also reduce the scar formation after surgery, but cytotoxicity and the administrative pathway prevent its application., Purpose: Our purpose was to design and attest the role of new delivery system MMC-PEG controlled-release film in preventing the epidural scar adhesions after laminectomy in the rat model., Study Design/setting: A total laminectomy of L1 in the rat model was used to assess epidural fibrosis between and around the spinal nerves using a histochemistry assessment along with flow cytometry analysis., Patient Sample: The sample comprises 64 adult male Sprague-Dawley rats., Outcome Measures: The outcome measures are macroscopic evaluation, histological analysis, and flow cytometry analysis., Methods: Lumbar laminectomies at L1 and L2 with a L1-L2 disc injury were performed on 64 adult male Sprague-Dawley rats. The rats were then randomized into four groups. In Group I, 25 mg PEG film was applied on the dura mater in the laminectomy area before the layers were sutured. In Group II, a cotton pad soaked with 0.05% MMC solution was kept on the laminectomy area for 5 minutes. In Group III, 25 mg PEG film containing 0.01% MMC was implanted on the laminectomy area. In Group IV, the laminectomy area was flushed with saline before wound closure. The rats were sacrificed 4 weeks after the operation. Macroscopic evaluation and histological analysis of epidural scar adhesion with the hematoxylin and eosin stain and Masson stain were used followed by the quantification of hydroxyproline (Hyp) and flow cytometry analysis of the apoptosis of fibroblasts in the scar tissues., Results: The recovery of all rats was uneventful after the operations. In the laminectomy sites of rats treated with MMC or MMC-PEG, the dura mater was clean without any evident adhesion or membrane. Collagen tissue hyperplasia significantly decreased in the MMC- or MMC-PEG-treated models. Accordingly, Hyp concentration was significantly reduced in these two groups compared with saline-control group. In addition, the apoptosis of fibroblasts, however, was significantly elevated in the MMC or MMC-PEG group compared with the saline-control group., Conclusions: These results demonstrate that the treatment of postlaminectomy wounds with MMC-PEG film reduces the severity of adhesion by decreasing the concentration of Hyp and increasing the apoptosis of fibroblasts., (Copyright 2010 Elsevier Inc. All rights reserved.)

Little is known about the influence of 10-hydroxycamptothecin (HCPT) on fibroblast proliferation and pathological changes in epidural scar tissue after laminectomy. Here we illustrated the effect of HCPT on fibroblast proliferation and epidural scar adhesion after laminectomy in rats. In the present study, seventy-two rats underwent laminectomies at Lumbar-1 level, then HCPT in various concentrations (0.1, 0.05, and 0.01 mg/ml) or saline (9 mg/ml) were applied to the laminectomy sites. Four weeks later the rats were killed and the epidural adhesion was evaluated. The area of epidural scar tissue and the number of fibroblasts were also determined. The degree of epidural adhesion was classified according to Rydell standard. The results showed that no or little epidural adhesions were seen in the laminectomy sites treated with 0.1 mg/ml HCPT. The Rydell classification, the area of epidural scar tissue and the number of fibroblasts in 0.1 mg/ml HCPT group were significantly less than those of 0.05 mg/ml HCPT group, 0.01 mg/ml HCPT group and saline group. Moderate epidural adhesions were noted in the laminectomy sites of 0.05 mg/ml HCPT group. The Rydell classification, the area of scar tissue and the number of fibroblasts were less than those of 0.01 mg/ml HCPT group and saline group. However, dense epidural adhesions were found in 0.01 mg/ml HCPT group and saline group. The Rydell classification, the area of scar tissue and the number of fibroblasts showed no significant difference compared with those of saline group. In conclusion, topical application of 0.1 mg/ml HCPT could effectively prevent fibroblast proliferation and reduce epidural adhesion after laminectomy in rats.

With a view to better understanding the role of oxidant/antioxidant variables in proliferation dynamics of somatic cells, we explored the relationships among superoxide dismutase (SOD) activity, glutathione peroxidase (Gpx) activity, reactive oxygen intermediates (ROI), and indices of cellular proliferation and senescence in cultured fibroblasts from Down syndrome and normal donors. We found that Down syndrome cells had a significantly slower proliferative rate, but attain replicative senescence at similar population doubling (PD) as control cells. Irrespective of donor origin, the number of PD until replicative senescence was positively correlated with Gpx activity (r = 0.784, P = 0.007). In addition, the presence of exogenous catalase in the growth medium significantly extended the number of PD until replicative senescence (P = 0.011). The loss of telomere repeats per PD was not different between Down syndrome cells and controls. However, SOD activity was inversely correlated with the loss of telomere repeats per PD. Collectively, these findings suggest that replicative senescence ultimately relates to mechanisms downstream to SOD (i.e., Gpx and catalase) and confirmed previous observations about inverse relationships between SOD activity and telomere repeat loss per cellular replication.

Background/Aims: Rheumatoid arthritis (RA) is a systemic chronic inflammatory disease characterised by prominent synoviocyte hyperplasia and a potential imbalance between the growth and death of fibroblast-like synoviocytes (FLS). Mitomycin C (MMC) has previously been demonstrated to inhibit fibroblast proliferation and to induce fibroblast apoptosis. However, the effects of MMC on the proliferation and apoptosis of human RA FLS and the potential mechanisms underlying its effects remain unknown. Methods: Cell viability was determined using the Cell Counting Kit-8 assay. Apoptotic cell death was analysed via Annexin V-FITC/PI double staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling. The production of intracellular reactive oxygen species (ROS) was assessed via flow cytometry, and the changes in mitochondrial membrane potential (ΔΨm) were visualized based on JC-1 staining via fluorescence microscopy. The expression of apoptosis-related proteins was determined via Western blot. Results: Treatment with MMC significantly reduced cell viability and induced apoptosis in RA FLS. Furthermore, MMC exposure was found to stimulate the production of ROS and to disrupt the ΔΨm compared to the control treatment. Moreover, MMC increased the release of mitochondrial cytochrome c, the ratio of Bax/Bcl-2, the activation of caspase-9 and caspase-3, and the subsequent cleavage of poly(ADP-ribose) polymerase. Conclusion: Our findings suggest that MMC inhibits cell proliferation and induces apoptosis in RA FLS, and the mechanism underlying this MMC-induced apoptosis may involve a mitochondrial signalling pathway. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) stress-mediated cell apoptosis has been implicated in various cell types, including fibroblasts. Previous studies have shown that mitomycin C (MMC)-induced apoptosis occurs in fibroblasts, but the effects of MMC on ER stress-mediated apoptosis in fibroblasts have not been examined. Here, MMC-induced apoptosis in human primary fibroblasts was investigated by exposing cells to a single dose of MMC for 5 minutes. Significant inhibition of cell proliferation and increased apoptosis were observed using a cell viability assay, Annexin V/propidium iodide double staining, cell cycle analysis, and TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining. Upregulation of proapoptotic factors, including cleaved caspase-3 and poly ADP-ribose polymerase (PARP), was detected by Western blotting. MMC-induced apoptosis was correlated with elevation of 78-kDa glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP), which are hallmarks of ER stress. Three unfolded protein response (UPR) sensors (inositol-requiring enzyme 1, IRE1; activating transcription factor 6, ATF6; and PKR-like ER kinase, PERK) and their downstream signaling pathways were also activated. Knockdown of CHOP attenuated MMC-induced apoptosis by increasing the ratio of BCL-2/BAX and decreasing BIM expression, suggesting that ER stress is involved in MMC-induced fibroblast apoptosis. Interestingly, knockdown of PERK significantly decreased ER stress-mediated apoptosis by reducing the expression of CHOP, BIM and cleaved caspase-3. Reactive oxygen species (ROS) scavenging also decreased the expression of GRP78, phospho-PERK, CHOP, and BIM. These results demonstrate that MMC-induced apoptosis is triggered by ROS generation and PERK activation. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to investigate the effects of topical application mitomycin-C (MMC) on wound healing after laminectomy. 60 adult male SD rats were equally and randomly divided into five groups. Laminectomy was performed at the level of L1 in all rats. After hemostasis was achieved, cotton pads soaked with saline and MMC (0.1mg/ml, 0.3mg/ml, 0.5mg/ml and 0.7mg/ml) were directly subjected to the exposed dura for 5min in each group. Two weeks after laminectomy all the rats were killed. The vertebral column including the back scar tissue and muscles was obtained to make paraffin sections. The hematoxylin–eosin staining and Masson staining were performed with the obtained paraffin sections. The number of the fibroblast and the capillary density were counted by the hematoxylin–eosin staining slice. The extent of epidural fibrosis and the expression of vascular endothelial growth factor (VEGF) were evaluated by the immunohistochemical slice through a computer image analysis system. Our data showed that the number of fibroblast, capillary density and fibrotic tissue in the 0.5 and 0.7mg/ml MMC groups was significantly lower than the control, 0.1 and 0.3mg/ml MMC groups; while the expression of VEGF in control and 0.1mg/ml MMC groups was notably higher than 0.3, 0.5 and 0.7mg/ml MMC groups. Topical application of MMC above the concentration of 0.3mg/ml could affect all steps of the wound healing process via inhibiting the angiogenesis and fibroblast proliferation, thus delayed the wound healing after laminectomy. [Copyright &y& Elsevier]