Is cultured tendon fibroblast a good model to study tendon healing?

Cultured tendon fibroblasts (CTFs) from intact explants are widely used to study tendon healing in vitro. The significance of these findings may rely on similarities between CTFs and healing tendon fibroblasts in situ. Our purpose was to compare CTFs with fibroblasts cultured from healing tendons. We cultured CTFs from intact and healing tendons at day 7 and day 14 postinjury in a rat model of patellar donor site injury. The mRNA expression of COL1A1, COL3A1, decorin, and biglycan, with or without supplementation of 1 ng/mL TGF‐β1, was compared by quantitative real‐time RT‐PCR. The expression of proliferation cell nuclear antigen (PCNA) and α‐smooth muscle actin (α‐SMA) was determined by immunostain. COL3A1 and decorin mRNA in CTFs was lower as compared to day 7 healing fibroblasts, but its biglycan mRNA level was higher than day 14 healing fibroblasts. TGF‐β1 increased COL1A1 and decorin mRNA in CTFs, but decreased the mRNA of all four genes in day 7 healing tendon fibroblasts. CTFs exhibited lower PCNA immunopositivity as compared to day 7 and day 14 healing fibroblasts, but a higher α‐SMA immunopositivity than cultured day 14 healing fibroblasts. These findings showed that CTFs did not resemble healing tendon cells with respect to major cellular activities related to tendon healing. Thus, fibroblasts from healing tendon may be a more appropriate model for studying cellular activities in tendon healing. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:374–383, 2008     

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.     

Expression of transforming growth factor β isoforms and their roles in tendon healing

Transforming growth factor β (TGF‐β) plays active roles in tendon healing. However, the differential effects of TGF‐β isoforms on tendon healing have not been investigated. In cultured tendon fibroblasts, we tested the effects of TGF‐β1, β2, and β3 on the mRNA levels of COL1A1 and COL3A1 by quantitative real‐time polymerase chain reaction. We also investigated the expression of TGF‐β isoforms, TGF‐β receptors, procollagen Type I and Type III in a rat model of tendon healing. We found that TGF‐β3 exhibited the highest potency in stimulating COL1A1 and COL3A1. TGF‐β1 exerted antagonistic effects to TGF‐β2 and β3. All TGF‐β isoforms and procollagen Type I were confined to the edges of the healing tendon at day 28 postinjury. Our results indicated that interaction of TGF‐β isoforms exist in the regulation of collagen synthesis in tendon fibroblasts. Their effects may be further complicated by uneven spatial distribution of TGF‐β and TGF‐β receptors in healing tendons.     

Triptolide attenuates renal interstitial fibrosis in rats with unilateral ureteral obstruction

Aim: Extracts of Tripterygium wilfordii Hook F. have been used to treat glomerulonephritis for more than 30 years in China. Most of the anti‐inflammatory and immunosuppressive activities of these extracts can be attributed to triptolide (Trip). The present study was to investigate the effect of Trip on renal interstitial fibrosis in a model of unilateral ureteral obstruction (UUO). Methods: UUO or sham‐operated rats were randomly assigned to receive mycophenolate mofetil (MMF), Trip or vehicle and were killed on days 7 and 14 after UUO or sham operation. Kidney specimens were fixed for immunohistochemistry for myofibroblasts (α‐smooth muscle actin, α‐SMA), macrophages (ED‐1), monocyte chemoattractant protein‐1 (MCP‐1) and osteopontin. Interstitial collagen deposition and amounts of transforming growth factor‐β1 (TGF‐β1) were determined by Sirius red staining and enzyme‐linked immunosorbent assay, respectively. The mRNA expression of TGF‐β1, connective tissue growth factor (CTGF), MCP‐1 and osteopontin were measured by real‐time polymerase chain reaction analysis. Results: The scores for the density of α‐SMA‐ and ED‐1‐positive cells, the staining of MCP‐1 and osteopontin, interstitial collagen deposition and amounts of TGF‐β1 were significantly reduced by MMF or Trip. MMF or Trip significantly reduced the mRNA expression of TGF‐β1, CTGF, MCP‐1 and osteopontin. Conclusion: Trip significantly attenuated tubulointerstitial fibrosis in a rat UUO model and the effect of Trip on renal fibrosis was similar to that of MMF. Trip may be useful as a potential candidate in the treatment of renal fibrosis.     

Nucleolin enhances the proliferation and migration of heat‐denatured human dermal fibroblasts

Denatured dermis, a part of dermis in burned skin, has the ability to restore its normal morphology and functions after their surrounding microenvironment is improved. However, the cellular and molecular mechanisms by which the denatured dermis could improve wound healing are still unclear. This study aimed to investigate the role of nucleolin during the recovery of heat‐denatured human dermal fibroblasts. Nucleolin mRNA and protein expression were significantly increased time‐dependently during the recovery of heat‐denatured human dermal fibroblasts (52 °C, 30 seconds). Heat‐denaturation promoted a time‐dependent cell proliferation, migration, chemotaxis, and scratched wound healing during the recovery of human dermal fibroblasts. These effects were prevented by knockdown of nucleolin expression with small interference RNA (siRNA), whereas overexpression of nucleolin enhanced cell proliferation, migration, and chemotaxis of human dermal fibroblasts with heat‐denaturation. In addition, the expression of transforming growth factor‐beta 1(TGF‐β1) was significantly increased during the recovery of heat‐denatured dermis and human dermal fibroblasts. TGF‐β1 expression was up‐regulated by nucleolin in human dermal fibroblasts. The results suggest that nucleolin expression is up‐regulated, and play an important role in promoting cell proliferation, migration, and chemotaxis of human dermal fibroblasts during the recovery of heat‐denatured dermis with a mechanism probably related to TGF‐β1.     

Altered TGF‐β signaling in fetal fibroblasts: What is known about the underlying mechanisms?

Scarless wound healing is a unique and intrinsic capacity of the fetal skin that is not fully understood. Further insight into the underlying mechanisms of fetal wound healing may lead to new therapeutic approaches promoting adult scarless wound healing. Differences between fetal and adult wound healing are found in the extracellular matrix, the inflammatory reaction and the levels of growth factors present in the wound. This review focuses specifically on transforming growth factor β (TGF‐β), as this growth factor is prominently involved in wound healing and fibroblast‐to‐myofibroblast differentiation. Although fetal fibroblasts do respond to TGF‐β, they lack a proliferative and a contractile response and display short‐lived myofibroblast differentiation, autocrine response, and collagen up‐regulation in comparison with adult fibroblasts. Curiously, prolonged TGF‐β activation is associated with fibrosis, and therefore, this short‐lived response in fetal fibroblasts might contribute to scarless healing. This review gives an overview of the current knowledge on TGF‐β signaling and the intracellular TGF‐β signaling pathway in fetal fibroblasts. Furthermore, this review also describes the various components that regulate the cellular TGF‐β response and hypothesizes about the possible roles these components might play in the altered response of fetal fibroblasts to TGF‐β.     

CCN5 Reduces Ligamentum Flavum Hypertrophy by Modulating the TGF‐β Pathway

Ligamentum flavum hypertrophy (LFH) is the most important component of lumbar spinal canal stenosis. Although the pathophysiology of LFH has been extensively studied, no method has been proposed to prevent or treat it. Since the transforming growth factor‐β (TGF‐β) pathway is known to be critical in LFH pathology, we investigated whether LFH could be prevented by blocking or modulating the TGF‐β mechanism. Human LF cells were used for the experiments. First, we created TGF‐β receptor 1 (TGFBR1) knock out (KO) cells with CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 biotechnology and treated them with TGF‐β1 to determine the effects of blocking the TGF‐β pathway. Subsequently, we studied the effect of CCN5, which has recently been proposed to modulate the TGF‐β pathway. To assess the predisposition toward fibrosis, α‐smooth muscle actin (αSMA), fibronectin, collagen‐1, collagen‐3, and CCN2 were evaluated with quantitative real‐time polymerase chain reaction, western blotting, and immunocytochemistry. The TGFBR1 KO LF cells were successfully constructed with high KO efficiency. In wild‐type (WT) cells, treatment with TGF‐β1 resulted in the overexpression of the messenger RNA (mRNA) of fibrosis‐related factors. However, in KO cells, the responses to TGF‐β1 stimulation were significantly lower. In addition, CCN5 and TGF‐β1 co‐treatment caused a notable reduction in mRNA expression levels compared with TGF‐β1 stimulation only. The αSMA protein expression increased with TGF‐β1 but decreased with CCN5 treatment. TGF‐β1 induced LF cell transdifferentiation from fibroblasts to myofibroblasts. However, this cell transition dramatically decreased in the presence of CCN5. In conclusion, CCN5 could prevent LFH by modulating the TGF‐β pathway. © 2019 The Authors. Journal of Orthopaedic Research^® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:2634–2644, 2019     

The effect of botulinum neurotoxin type A on capsule formation around silicone implants: the in vivo and in vitro study

This study confirms that botulinum neurotoxin type A (BoNT‐A) decreases capsular contracture and elucidates a possible mechanism. Silicone blocks were implanted subcutaneously in 20 mice. The experimental groups received BoNT‐A (1, 2·5 or 5 U) instilled into the subcutaneous pocket. After 30 days, periprosthetic capsules were harvested and evaluated. The effect of BoNT‐A on the differentiation of human dermal fibroblasts to myofibroblasts in culture was examined by Western blot analysis. Changes in transforming growth factor‐beta1 (TGF‐β1) expression in cultured fibroblasts were determined by enzyme‐linked immunosorbent assay (ELISA). In in vivo study, the thickness of capsules (P < 0·05) and the number of alpha‐smooth muscle actin (α‐SMA)⁺ cells in capsules (P < 0·05) were significantly decreased in the experimental groups. TGF‐β1 was significantly underexpressed in the experimental groups (P < 0·05). In in vitro study, BoNT‐A did not significantly affect fibroblast viability. Western blot analysis showed that α‐SMA protein levels were significantly decreased in the experimental groups (P < 0·05). Based on ELISA, the amount of TGF‐β1 was significantly decreased in the experimental groups (P < 0·05), especially cells treated with a high dose of BoNT‐A (P < 0·001). This study confirms that BoNT‐A prevents capsular formation around silicone implants, possibly by blocking TGF‐β1 signalling and interrupting the differentiation of fibroblasts to myofibroblasts.     

GDF‐5/7 and bFGF activate integrin α2‐mediated cellular migration in rabbit ligament fibroblasts

Cellular activities responding to growth factors are important in ligament healing. The anterior cruciate ligament (ACL) has poor healing potential compared to the medial collateral ligament (MCL). To assess the differences, we investigated the proliferation, migration, adhesion, and matrix synthesis responding to growth factors in rabbit ACL and MCL fibroblasts. ACL cell proliferation to basic fibroblast growth factor (bFGF), bone morphogenetic protein‐2, growth and differentiation factor (GDF)‐5, and GDF‐7 treatment was similar to that of MCL cells. GDF‐5 enhanced Col1a1 expression in ACL and MCL fibroblasts up to 4.7‐ and 17‐fold levels of control, respectively. MCL fibroblasts showed stronger migration activities in response to bFGF and GDF‐5 than ACL cells. GDF‐5/7 and bFGF also changed the stress fiber formation and cellular adhesion by modulating the distribution of integrin α2. Functional blocking analyses using anti‐integrin α2 antibodies revealed that cellular migration responding to growth factors depended on the integrin α2‐mediated adhesion on type I collagen. The expression of integrin α2 was also increased by growth factors in both cells. Our results demonstrate that GDF‐5/7 and bFGF stimulate cellular migration by modulating integrin α2 expression and integrin α2‐dependent adhesion, especially in MCL fibroblasts. These findings suggest that the different healing potential between ACL and MCL may be caused by different cellular behavior in the integrin α2‐mediated cellular migration in response to growth factors. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:225–231, 2010     

Rosiglitazone treatment attenuates renal tissue inflammation generated by urinary tract obstruction

Aim: Peroxisome proliferator‐activated receptor (PPAR)‐γ activation by rosiglitazone decreases manifestation of intrarenal inflammatory hallmarks. Inflammation significantly aggravates renal injury following urinary tract obstruction. The effect of rosiglitazone on renal inflammation following unilateral ureteral obstruction was investigated. Methods: Ninety‐six Srague–Dawley rats were subjected to unilateral ureteral ligation, or to sham operation. Half of each group received rosiglitazone, 5 mg/kg bodyweight per day. The animals were killed and their kidneys allocated following 1 h, 24 h or 2 weeks, for pathological examination or for intrarenal transforming growth factor (TGF)‐β, interleukin (IL)‐4, IL‐6, IL‐10 and nitric oxide (NO) assessment by specific enzyme‐linked immunosorbent assays. Apoptosis rates, extracellular matrix deposition, PPAR‐γ, α‐smooth muscle actin (α‐SMA) expression and macrophage infiltration were assessed by specific immunohistological stainings. Results: PPAR‐γ receptor expression was downregulated, and infiltration of macrophages decreased, in all rosiglitazone‐treated kidneys. Rosiglitazone significantly decreased apoptosis, TGF‐β, IL‐6, α‐SMA expression and NO availability in obstructed kidneys. Synthesis of IL‐10 was unaltered, while IL‐4 augmented by Rosiglitazone. Rosiglitazone also affected NO and IL‐4 production in sham‐operated controls. Conclusion: (i) Rosiglitazone attenuates profibrotic and pro‐inflammatory responses in a rat model of ureteral obstruction‐induced renal inflammation; (ii) rosiglitazone stimulates counteractive anti‐inflammatory responses in the damaged kidneys; (iii) in part, rosiglitazone exerts comparable anti‐inflammatory effects on obstructed kidneys and unobstructed healthy controls. Taken together, this ascertains the importance of the anti‐inflammatory role of rosiglitazone treatment in amelioration of ureteral obstruction‐induced renal damage.     

Wnt pathway regulation by demineralized bone is approximated by both BMP‐2 and TGF‐β1 signaling

Allogeneic demineralized bone is used extensively as a clinical graft material because it has osteo/chondroinductive and osteoconductive properties. Demineralized bone powder (DBP) induces chondrogenic differentiation of human dermal fibroblasts (hDFs) in three‐dimensional collagen cultures, but the initiating mechanisms have not been fully characterized nor has it been shown that bone morphogenetic proteins (BMPs) recapitulate DBP's effects on target cells. Among the many signaling pathways regulated in hDFs by DBP prior to in vitro chondrogenesis, there are changes in Wnts and their receptors that may contribute to DBP actions. This study tests the hypothesis that DBP modulation of Wnt signaling entails both BMP and TGF‐β pathways. We compared the effects of DBP, TGF‐β1, or BMP‐2 on Wnt signaling components in hDFs by Wnt signaling macroarray, RT‐PCR, in situ hybridization, and Western immunoblot analyses. Many effects of DBP on Wnt signaling components were not shared by BMP‐2, and likewise DBP effects on Wnt genes and β‐catenin only partially required the TGF‐β pathway, as shown by selective inhibition of TGF‐β/activin receptor‐like kinase. The analyses revealed that 64% (16/25) of the Wnt signaling components regulated by DBP were regulated similarly by the sum of effects by BMP‐2 and by TGF‐β1. In conclusion, signaling mechanisms of inductive DBP in human dermal fibroblasts involve the modulation of multiple Wnt signals through both BMP and TGF‐β pathways. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 554–560, 2013     

Effects of proteasome inhibitors on rat renal fibrosis in vitro and in vivo

Aim: Transforming growth factor‐β (TGF‐β) is involved in renal tubulointerstitial fibrosis. Recently, the ubiquitin proteasome system was shown to participate in the TGF‐β signalling pathway. The aim of this study was to examine the effects of proteasome inhibitors on TGF‐β‐induced transformation of renal fibroblasts and tubular epithelial cells in vitro and on unilateral ureteral obstruction (UUO) in vivo. Methods: Rat renal fibroblasts NRK‐49F cells and tubular epithelial cells, NRK‐52E, were treated with TGF‐β in the presence or absence of a proteasome inhibitor, MG132 or lactacystin. Rats were subjected to UUO and received MG132 i.p. for 7 days. Results: In cultured renal cells, both MG132 and lactacystin inhibited TGF‐β‐induced α‐smooth muscle actin (α‐SMA) protein expression according to both western blotting and immunofluorescent study results. MG132 also suppressed TGF‐β‐induced mRNA expression of α‐SMA and upregulation of Smad‐response element reporter activity. However, MG132 did not inhibit TGF‐β‐induced phosphorylation and nuclear translocation of Smad2. In contrast, MG132 increased the protein level of Smad co‐repressor SnoN, demonstrating that SnoN is one of the target molecules by which MG132 blocks the TGF‐β signalling pathway. Although the proteasome inhibitor suppressed TGF‐β‐induced transformation of cultured fibroblasts and tubular epithelial cells, MG132 treatment did not ameliorate tubulointerstitial fibrosis in the rat UUO model. Conclusion: Proteasome inhibitors attenuate TGF‐β signalling by blocking Smad signal transduction in vitro, but do not inhibit renal interstitial fibrosis in vivo.     

Nox2 is a Mediator of Chronic CsA Nephrotoxicity

We hypothesized that Nox2, the classical phagocytic NADPH oxidase, plays an important role in calcineurin inhibitor (CNI)‐induced renal fibrosis. We tested this hypothesis in vitro, in animal and in human studies. Cyclosporine A (CsA) and tacrolimus (TAC) were associated with greater levels of Nox2 mRNA and epithelial to mesenchymal transition (EMT) in NRK52E cells. CsA increased Nox2, α‐SMA and phosphorylated‐p38MAPK, Smad3 and NFκB proteins. Nox2 upregulation and EMT were inhibited in TGF‐β1 knockout cells suggesting that TGF‐β1 is required for Nox2 activation. Fisher344 rats treated with high dose CsA showed increased Nox2 in the tubulointerstitium and greater Nox2, α‐SMA, phosphorylated Smad3 and nitrotyrosine by immunoblot analyses. Inhibition of Nox2 by coadministration of apocynin or diphenyleneiodonium was associated with reduced fibrogenesis. We validated these findings by treating wild type and Nox2 null (B6.129S‐Cybb^Tm1Din/J) mice with high dose CsA. Western blot analyses confirmed the absence of Nox2 and significantly lower levels of α‐SMA and 4‐hydroxynonenal (HNE) in CsA‐treated knockout mice. These findings were clinically relevant since Nox2 and α‐SMA were increased in the tubulointerstitium of kidneys from 15 liver transplant recipients with biopsy‐confirmed chronic CsA or TAC nephrotoxicity. In conclusion, specific Nox2 inhibition strategies may improve chronic CNI nephrotoxicity in solid organ transplantation.     

Erythropoietin and its non‐erythropoietic derivative: Do they ameliorate renal tubulointerstitial injury in ureteral obstruction?

Objectives: Pleiotropic effects of recombinant human erythropoietin (EPO) have recently been discovered in many non‐renal animal models. The renoprotective effects of EPO and carbamylated‐erythropoietin (CEPO), a novel EPO which has a small stimulatory effect on hemoglobin, have never been explored in unilateral ureteral obstruction (UUO), a chronic tubulointerstitial (TI) disease model which is independent of systemic factors. Methods: In order to examine the effects of EPO and CEPO treatments on renal TI injury, 36 male Sprague‐Dawley rats, weighing 250–320 g, underwent: UUO without treatment (group 1, n = 12), UUO with EPO (groups 2, n = 12), and UUO with CEPO (group 3, n = 12). EPO and CEPO were injected subcutaneously at a dose of 5000 u/kg to each respective rat at 1 day pre‐UUO and at day 3, 7 and 10 post‐UUO. After days 3, 7, and 14 of UUO, TI injury, collagen, α‐smooth muscle actin (α‐SMA) positive cell, ED1‐positive cell, terminal deoxynucleotidyl transferase (TdT) mediated nick‐end labeling (TUNEL)‐positive cell, and transforming growth factor‐β1 (TGF‐β1) messenger ribonucleic acid (mRNA) were determined. Bcl‐2 expression was also assessed to verify the mechanism of apoptosis. Results: At day 14 UUO caused severe TI injury with a significant increase in collagen, α‐SMA, ED1‐positive cell, TUNEL‐positive cell, and TGF‐β1 mRNA expression. Administration of EPO and CEPO significantly attenuated TI injury, collagen, ED1‐positive cells, and TUNEL‐positive cells. Only CEPO‐treated rats had decreased α‐SMA positive cells and TGF‐β1 mRNA. The expression of Bcl‐2 was demonstrated only in EPO‐treated rats. The hematocrit levels in EPO‐treated rats were higher than the control and CEPO‐treated rats. Conclusions: EPO and CEPO can limit 14‐day UUO‐induced TI injury by reducing inflammation, interstitial fibrosis, and tubular apoptosis.     

Matrix metalloproteinase inhibition delays wound healing and blocks the latent transforming growth factor‐β1‐promoted myofibroblast formation and function

The ability to regulate wound contraction is critical for wound healing as well as for pathological contractures. Matrix metalloproteinases (MMPs) have been demonstrated to be obligatory for normal wound healing. This study examined the effect that the broad‐spectrum MMP inhibitor BB‐94 has when applied topically to full‐thickness skin excisional wounds in rats and its ability to inhibit the promotion of myofibroblast formation and function by the latent transforming‐growth factor‐β1 (TGF‐β1). BB‐94 delayed wound contraction, as well as all other associated aspects of wound healing examined, including myofibroblast formation, stromal cell proliferation, blood vessel formation, and epithelial wound coverage. Interestingly, BB‐94 dramatically increased the level of latent and active MMP‐9. The increased levels of active MMP‐9 may eventually overcome the ability of BB‐94 to inhibit this MMP and may explain why wound contraction and other associated events of wound healing were only delayed and not completely inhibited. BB‐94 was also found to inhibit the ability of latent TGF‐β1 to promote the formation and function of myofibroblasts. These results suggest that BB‐94 could delay wound closure through a twofold mechanism; by blocking keratinocyte migration and thereby blocking the necessary keratinocyte–fibroblast interactions needed for myofibroblast formation and by inhibiting the activation of latent TGF‐β1.     

Inflammation and Epithelial to Mesenchymal Transition in Lung Transplant Recipients: Role in Dysregulated Epithelial Wound Repair

Epithelial to mesenchymal transition (EMT) has been implicated in the pathogenesis of obliterative bronchiolitis (OB) after lung transplant. Although TNF‐α accentuates TGF‐β1 driven EMT in primary human bronchial epithelial cells (PBECs), we hypothesized that other acute pro‐inflammatory cytokines elevated in the airways of patients with OB may also accentuate EMT and contribute to dysregulated epithelial wound repair. PBECs from lung transplant recipients were stimulated with TGF‐β1 ± IL‐1β, IL‐8, TNF‐α or activated macrophages in co‐culture and EMT assessed. The quality and rate of wound closure in a standardized model of lung epithelial injury was assessed in response to above stimuli. Co‐treatment with TGF‐β1 + TNF‐α or IL‐1β significantly accentuates phenotypic and some functional features of EMT compared to TGF‐β1 alone. Co‐treatment with TGF‐β1 + TNF‐α or IL‐1β accelerates epithelial wound closure however the quality of repair is highly dysregulated. Co‐treatment with TGF‐β1 + IL‐8 has no significant effect on EMT or the speed or quality of wound healing. Activated macrophages dramatically accentuate TGF‐β1‐driven EMT and cause dysregulated wound repair. Crosstalk between macrophage‐derived acute inflammation in the airway and elevated TGF‐β1 may favor dysregulated airway epithelial repair and fibrosis in the lung allograft via EMT.     

Intrinsic healing response of the human anterior cruciate ligament: An histological study of reattached ACL remnants

A reattachment of the tibial remnant of the torn anterior cruciate ligament (ACL) to the posterior cruciate ligament is sometimes observed during surgery and apparently implies that the human ACL does have a healing response. The aim of this study was to investigate whether this reattachment tissue has similar histological characteristics of a healing response as the medial collateral ligament (MCL), which can heal spontaneously. Standard histology and immunostaining of α‐smooth muscle actin and collagen type 3 was performed. The results shows that the reattached tissue has typical characteristics of a healing response: the reattached ACL remnant could not be released by forceful traction; microscopy showed that the collagen fibers of the reattached tissue were disorganized with no preferred direction; increased neovascularization; the presence of lipid vacuoles; the mean number of cells within the biopsy tissue was 631 ± 269 cells per mm²; and 68 ± 20% was expressing α‐SMA; semi‐quantitative analysis of collagen type 3 expression showed that collagen type 3 had an high expression with an average score of 3. In conclusion, this study shows that the human proximal 1/3 ACL has an intrinsic healing response with typical histological characteristics similar to the MCL. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:296–301, 2014.     

Transforming growth factor‐β1 suppresses the up‐regulation of matrix metalloproteinase‐2 by lung fibroblasts in response to tumor necrosis factor‐α

Exposed to inflammatory factors or cytokines, fibroblasts appear to play additional roles beyond the deposition of extracellular matrix. It has been reported that tumor necrosis factor‐α (TNF‐α) induces the production of matrix metalloproteinase‐2 (MMP‐2) and transforming growth factor‐β1 (TGF‐β1) in fibroblasts. In this study, we demonstrated that the active MMP‐2 secreted by lung fibroblasts reached the peak level at 12 hours after TNF‐α treatment, whereas, by adding anti‐TGF‐β1 antibody in the culture medium, the MMP‐2 production in response to TNF‐α was maintained at high levels after 24 hours of treatment. We also confirmed that TNF‐α induced up‐regulation of active TGF‐β1 and exogenous TGF‐β1 induced down‐regulation of MMP‐2 synthesis in lung fibroblasts. Moreover, an increased MMP‐2 level was observed in a rat model with pulmonary inflammation and fibrosis induced by bleomycin‐A5. This revealed that MMP‐2 in the lung reached the peak level when TNF‐α reached the peak level at the 7th day, and then MMP‐2 decreased along with an increase in the TGF‐β1 level. Taken together, our results demonstrate that TNF‐α induced an increase of MMP‐2 and TGF‐β1 in lung fibroblasts, and the TGF‐β1 attenuated the up‐regulation of MMP‐2. This suggests that MMP‐2 secreted from fibroblasts modulated by TNF‐α/TGF‐β1 might play an important role in pulmonary inflammation and fibrosis.     

Differential MMP-2 activity of ligament cells under mechanical stretch injury: an in vitro study on human ACL and MCL fibroblasts.

Recent studies have revealed that following injuries, ligament tissues such as anterior cruciate ligaments (ACL), release large amounts of matrix metalloproteinases (MMPs). These enzymes have a devastating effect on the healing process of the injured ligaments. Although these enzymes are produced following ligament injuries, because of different healing capacities seen between the medial collateral ligament (MCL) and ACL, we were curious to find if the MMP activity was expressed and modulated differently in these tissues. For this purpose ACL and MCL fibroblasts were seeded on equi-biaxial stretch chambers and were stretched in different levels. The stretched cells were assayed using Zymography, Western Blot and global MMP activity assays. The results showed that within 72 h after injurious stretch, production of 72 kD pro-MMP-2 increased in both ACL and MCL. However, the ACL fibroblasts generated significantly more pro-MMP-2 than the MCL fibroblasts. Furthermore we found in ACL pro-MMP-2 was converted more into active form. With 4-aminophenyl mercuric acetate (APMA) treatment, large amounts of pro-MMP-2 were converted into active form in both. This indicates that there is no significant difference between ACL and MCL fibroblasts in post-translational modification of MMP-2. The fluorescent MMP activity assays revealed that the MMP family activities were higher in the injured ACL fibroblasts than the MCL. Since the MMPs are critically involved in extracellular matrix (ECM) turnover, these findings may explain one of the reasons why the injured ACL hardly repairs. The higher levels of active MMP-2 seen in the ACL injuries may disrupt the delicate balance of ECM remodeling process. These results suggest that the generation and modulation of MMP-2 may be directly involved in the different responses seen in ACL and MCL injuries.