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.     

Age dependence of expression of growth factor receptors in porcine ACL fibroblasts

Tissue engineering approaches that harness the stimulatory power of platelet‐rich plasma have produced encouraging results in anterior cruciate ligament (ACL) repair. However, a number of recent studies have demonstrated age‐dependent differences in cellular responses to such an approach. Identifying the reasons for these differences would allow counteracting them and consequently improve outcomes. In this study we hypothesized that these age‐related effects are caused by differences in the expression of the receptors for growth factors released from platelet‐rich plasma (PRP). Porcine ACL fibroblasts from a predetermined number of animals of different ages were obtained, and mRNA levels of the receptors of platelet‐derived growth factor (PDGF), transforming growth factor β (TGF‐β), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF) were determined. Expression levels were compared across age groups (young and adolescent) and regressed on age in days. While no significant difference was seen across groups, the regression analysis showed decreases in receptor expression with increasing age. These differences were statistically significant for TGF‐β receptor 1, FGF receptor, and VEGF receptor 2; and borderline significant for TGF‐β receptor 3 and PDGF receptor. The only receptor that was not associated with age was VEGF receptor 1, a regulator of VEGF receptor 2. These findings suggest that the decrease in growth factor receptor expression as a likely reason for reduced PRP action with increasing age. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1107–1112, 2010     

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.     

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.     

Hyaluronic acid modulates gene expression of connective tissue growth factor (CTGF), transforming growth factor‐β1 (TGF‐β1), and vascular endothelial growth factor (VEGF) in human fibroblast‐like synovial cells from advanced‐stage osteoarthritis in vitro

Intraarticular injection of hyaluronan (hyaluronic acid; HA) is the common way to treat osteoarthritis (OA) of knees. This treatment cannot only maintain the viscoelastic properties of knee but also release the OA pain. However, the exact molecular mechanism is unknown. In this study, after human synovial cells were stimulated with HA and Hylan (Synvisc®) for 24 h, real‐time polymerase chain reaction (real‐time PCR) was used to detect the alteration of connective tissue growth factor (CTGF), transforming growth factor‐β1 (TGF‐β1), and vascular endothelial growth factor (VEGF) gene expression, which were specific genes related to pathogenesis of OA knees. Our results illustrated that both HA and Hylan might not cause cytotoxicity or apoptosis of synovial cells in serum deprivation environment. The gene expressions of TGF‐β1 and VEGF were significantly increased at the concentration of 0.1 mg/mL HA and 0.1 mg/mL Hylan, respectively (α < 0.05). The synovial cells with treatment of 0.1 mg/mL Hylan decreased the CTGF gene expression (0.66‐fold) and VEGF (0.78‐fold) compared to 0.1 mg/mL HA (α < 0.05). We suggested that the profile of CTGF, TGF‐β1, and VEGF gene expressions in our study might provide the rational mechanism for the therapeutic effect of hyaluronan on OA knees. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:492–496, 2010     

Temporal growth factor release from platelet‐rich plasma,trehalose lyophilized platelets,and bone marrow aspirate and their effect on tendon and ligament gene expression

Platelet‐rich plasma (PRP) has generated substantial interest for tendon and ligament regeneration because of the high concentrations of growth factors in platelet α‐granules. This study compared the temporal release of growth factors from bone marrow aspirate (BMA), PRP, and lyophilized platelet product (PP), and measured their effects on tendon and ligament gene expression. Blood and BMA were collected and processed to yield PRP and plasma. Flexor digitorum superficialis tendon (FDS) and suspensory ligament (SL) explants were cultured in 10% plasma in DMEM (control), BMA, PRP, or PP. TGF‐β1 and PDGF‐BB concentrations were determined at 0, 24, and 96 h of culture using ELISA. Quantitative RT‐PCR for collagen types I and III (COL1A1, COL3A1), cartilage oligomeric matrix protein (COMP), decorin, and matrix metalloproteinases‐3 and 13 (MMP‐3, MMP‐13) was performed. TGF‐β1 and PDGF‐BB concentrations were highest in PRP and PP. Growth factor quantity was unchanged in BMA, increased in PRP, and decreased in PP over 4 days. TGF‐β1 and platelet concentrations were positively correlated. Lyophilized PP and PRP resulted in increased COL1A1:COL3A1 ratio, increased COMP, and decreased MMP‐13 expression. BMA resulted in decreased COMP and increased MMP‐3 and MMP‐13 gene expression. Platelet concentration was positively correlated with COL1A1, ratio of COL1A1:COL3A1, and COMP, and negatively correlated with COL3A1, MMP‐13, and MMP‐3. White blood cell concentration was positively correlated with COL3A1, MMP3, and MMP13, and negatively correlated with a ratio of COL1A1:COL3A1, COMP, and decorin. These findings support further in vivo investigation of PRP and PP for treatment of tendonitis and desmitis. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 1033–1042, 2009     

水蛭素对皮肤增生性瘢痕成纤维细胞bFGF及TGFβ_1表达的影响

目的：检测水蛭素对人皮肤瘢痕成纤维细胞碱性成纤维细胞因子（basic fibroblast growth factor,bFGF）、转化生长因子β1（transforming growth factor beta 1,TGFβ1）表达的影响,探讨水蛭素抑制瘢痕形成的作用及机制。方法：体外培养并鉴定人皮肤瘢痕成纤维细胞,实时荧光定量RT-PCR和免疫细胞化学法分别检测不同浓度水蛭素作用人成纤维细胞24h后,bFGF、TGFβ1的mRNA和bFGF、TGFβ1蛋白表达水平。结果：浓度为0.156～2.5 U/L的水蛭素可下调瘢痕成纤维细胞TGFβ1mRNA、蛋白的表达,同时上调bFGF的mRNA、蛋白的表达,不同浓度组间比较,差异有统计学意义。水蛭素可抑制增生性瘢痕成纤维细胞分泌TGFβ1,促进其分泌bFGF。结论：水蛭素抑制瘢痕可调节bFGF、TGFβ1分泌,由此抑制成纤维细胞的生长、增殖并进一步抑制瘢痕形成。     

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.     

Relationship of cytokine levels and clinical effect on platelet‐rich plasma‐treated lateral epicondylitis

Lateral epicondylitis (LE) is difficult to manage and can result in significant patient morbidity. Currently, the clinical use of platelet‐rich plasma (PRP) for painful tendons has received attention, but its efficacy remains controversial. This study aimed to investigate the clinical effects of PRP and its biological components. A total of 156 patients with LE were randomly divided into group 1, treated with a single injection of 2‐ml autologous PRP, and group 2, treated with a control received only physical therapy without injection. Both groups used a tennis elbow strap and performed stretching and strengthening exercises during 24 weeks’ follow‐up. Pain and functional improvements were assessed using the visual analog scale (VAS), Modified Mayo Clinic Performance Index for the elbow, and magnetic resonance imaging (MRI). White blood cell count, platelet count, and levels of platelet‐derived growth factor‐AB (PDGF‐AB), PDGF‐BB, transforming growth factor‐β (TGF‐β), vascular endothelial growth factor, epithelial growth factor, and interleukin‐1 β in PRP were measured and investigated for statistical correlation with the clinical score. At 24 weeks, all pain and functional variables, including VAS score, Mayo Clinic performance scores, and MRI grade, improved significantly in group 1 (p < 0.05). PDGF‐AB, PDGF‐BB, and TGF‐β levels were more significantly increased in PRP than in whole blood. TGF‐β level significantly correlated with Mayo Clinic performance score and MRI grade improvement. Thus, TGF‐β level in PRP is considered to play a pivotal role in tendon healing. These results may contribute to identifying the best protocol for PRP application in tendinopathies. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:913–920, 2018.

     

Transforming growth factor β1 regulates the expression of CCN2 in human keratinocytes via Smad‐ERK signalling

Connective tissue growth factor (CCN2/CTGF) and transforming growth factor β1 (TGF‐β1) are important regulators of skin wound healing, but controversy remains regarding their expression in epithelial cell lineages. Here, we investigate the expression of CCN2 in keratinocytes during reepithelialisation and its regulation by TGF‐β1. CCN2 was detected in the epidermis of healing full‐thickness porcine wounds. Human keratinocytes were incubated with or without 10 ng/ml TGF‐β1, and signalling pathways were blocked with 10‐μM SIS3 or 20‐μM PD98059. Semi‐quantitative real‐time PCR was used to study CCN2 mRNA expression, and western blot was used to measure CCN2, phosphorylated‐ERK1/2, ERK1/2, phosphorylated‐Smad3 and Smad2/3 proteins. CCN2 was transiently expressed in neoepidermis at the leading edge of the wound in vivo. In vitro, CCN2 expression was induced by TGF‐β1 at 2 hours (7·5 ± 1·9‐fold mRNA increase and 3·0 ± 0·6‐fold protein increase) and 12 hours (5·4 ± 1·9‐fold mRNA increase and 3·3 ± 0·6‐fold protein increase). Compared with inhibiting the SMAD pathway, inhibiting the mitogen‐activated protein kinase (MAPK) pathway was more effective in reducing TGF‐β1‐induced CCN2 mRNA and protein expression. Inhibition of the MAPK pathway had minimal impact on the activity of the SMAD pathway. CCN2 is expressed in keratinocytes in response to tissue injury or TGF‐β1. In addition, TGF‐β1 induces CCN2 expression in keratinocytes through the ras/MEK/ERK pathway. A complete understanding of CCN2 expression in keratinocytes is critical to developing novel therapies for wound healing and cutaneous malignancy.     

Endothelial cells incubated with platelet‐rich plasma express PDGF‐B and ICAM‐1 and induce bone marrow stromal cell migration

Platelet‐rich plasma (PRP) is used to accelerate bone repair through the growth factors released by platelets. The purpose of this study was to evaluate if PRP induce human umbilical vein endothelial cells (HUVEC) to express mRNA for osteogenic growth factors and stimulate the migration of bone marrow stromal cell (BMSC). The effects of PRP were compared to those induced by vascular endothelial growth factor‐A (VEGF‐A) or, as a negative control, by platelet poor plasma (PPP). After incubation with PRP, but not with PPP, HUVEC showed an increased expression of mRNA for platelet derived growth factor‐B (PDGF‐B), and this effect was not inhibited by an anti‐VEGF‐A antibody. The migration of BMSC was more stimulated by HUVEC incubated with PRP than by HUVEC incubated with low serum medium or PPP. Besides, PRP increased the expression of intercellular adhesion molecule‐1 (ICAM‐1) and osteoprotegerin, but did not affect the expression either of the receptor activator for nuclear factor κB ligand (RANKL) or of RANK. These findings support the hypothesis that PRP contribute to bone repair by favoring the pro‐osteogenic function of endothelial cells, including the recruitment of osteoblast precursors and the expression of adhesion molecules for monocyte/macrophages, while inhibiting their pro‐osteolytic properties. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1493–1498, 2009     

Transforming growth factor‐β (TGF‐β) expression is increased in the subsynovial connective tissues of patients with idiopathic carpal tunnel syndrome

Non‐inflammatory fibrosis of the subsynovial connective tissue (SSCT) is a hallmark of carpal tunnel syndrome (CTS). The etiology of this finding and its relationship to the development of CTS remain poorly understood. Recent studies have found that transforming growth factor‐β (TGF‐β) plays a central role in fibrosis. The purpose of this study was to investigate the expression of TGF‐β and connective tissue growth factor (CTGF), a downstream mediator of TGF‐β, in the pathogenesis of CTS. We compared SSCT specimens from 26 idiopathic CTS patients with specimens from 10 human cadaver controls with no previous diagnosis of CTS. Immunohistochemistry was performed to determine levels TGF‐β1, CTGF, collagen 1(Col1) and collagen 3 (Col3) expression. TGF‐β1 (p < 0.01), CTGF (p < 0.01), and Col3 (p < 0.01) were increased in SSCT of CTS patients compared with control tissue. In addition, a strong positive correlation was found between TGF‐β1 and CTGF, (R² = 0.80, p < 0.01) and a moderate positive correlation between Col3 and TGF‐β1 (R² = 0.49, p < 0.01). These finding suggest that there is an increased expression of TGF‐β and CTGF, a TGF‐β regulated protein, and that this TGF‐β activation may be responsible for SSCT fibrosis in CTS patients. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:116–122, 2014.     

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.     

A Dermal Equivalent Engineered with TGF‐β3 Expressing Bone Marrow Stromal Cells and Amniotic Membrane: Cosmetic Healing of Full‐Thickness Skin Wounds in Rats

Transforming growth factor beta‐3 (TGF‐β3) has been shown to decrease scar formation after scheduled topical applications to the cutaneous wounds. This study aimed to continuously deliver TGF‐β3, during the early phase of wound healing, by engineering a dermal equivalent (DE) using TGF‐β3 expressing bone marrow stromal cells (BM‐SCs) and human dehydrated amniotic membrane (hDAM). To engineer a DE, rat BM‐SCs were seeded on the hDAM and TGF‐β3 was transiently transfected into the BM‐SCs using a plasmid vector. Pieces of the dermal equivalent were transplanted onto the full‐thickness excisional skin wounds in rats. The process of wound healing was assessed by image analysis, Manchester Scar Scale (MSS), and histopathological studies 7, 14, 21, and 85 days after the excision. The results confirmed accurate construction of recombinant pcDNA3.1‐TGF‐β3 expression system and showed that the transfected BM‐SCs seeded on hDAM expressed TGF‐β3 mRNA and protein from day 3 through day 7 after transfection. After implantation of the DE, contraction of the wounds was measured from day 7 through 21 and analyzed by linear regression, which revealed that the rate of wound contraction in all experimental groups was similar. Histologic evaluation demonstrated that transfected BM‐SCs decreased retention and recruitment of the cells during the early stage of wound healing, decreased the formation of vascular structures and led to formation of uniformly parallel collagen bundles. MSS scores showed that TGF‐β3 secreting cells significantly improved the cosmetic appearance of the healed skin and decreased the scar formation. From these results, it could be concluded that transient secretion of TGF‐β3, during the early phase of healing, by BM‐SCs seeded on hDAM can improve the cosmetic appearance of the scar in cutaneous wounds without negatively affecting the process of wound repair.     

药物抑制兔耳病理性瘢痕中结缔组织生长因子的实验

目的探讨能有效抑制结缔组织生长因子（CTGF）的药物,以便为治疗病理性瘢痕提供依据。方法选择24只大耳白兔建立兔耳病理性瘢痕模型,随机分为4组：A组注射CTGF反义寡核苷酸,B组注射复方倍他米松,C组注射醋酸曲安奈得,D组为对照组,仅注射生理盐水。通过原位杂交法分别检测瘢痕组织中不同治疗组不同时段的CTGF表达,并通过苏木精-伊红（HE）染色法检测瘢痕组织中不同治疗组不同时段的成纤维细胞数。结果A、B、C3组在同一时段其CTGF表达均比D组低,差异有统计学意义（P〈0．05）,A组较B、C两组CTGF表达低,差异也具有统计学意义（P〈0．05）。B、C两组之间CTGF表达差异无统计学意义（P〉0．05）。成纤维细胞计数结果与以上结果基本一致。结论CTGF反义寡核苷酸、复方倍他米松、醋酸曲安奈得均可抑制病理性瘢痕中CTGF的表达,CTGF反义寡核苷酸抑制效果最明显。