Sircol Collagen Assay Research Articles

Epigallocatechin‐3‐Gallate Inhibits Collagen Production of Nasal Polyp‐Derived Fibroblasts

Nasal polyps are chronic inflammatory conditions characterized by myofibroblast differentiation and extracelluar matrix accumulation. The major catechin from green tea is (-)-epigallocatechin-3-gallate (EGCG), which has garnered attention for its potential to prevent oxidative stress-related diseases. The purpose of this study was twofold: (i) to determine the effect of EGCG on fibroblast differentiation into myofibroblasts and extracellular matrix accumulation in transforming growth factor (TGF)-β1-induced nasal polyp-derived fibroblasts (NPDFs) and (ii) to determine if the antioxidative effect of EGCG on reactive oxygen species (ROS) production in TGF-β1-induced NPDFs is involved in the aforementioned processes. TGF-β1-induced NPDFs were treated with or without EGCG. α-smooth muscle actin (α-SMA) and collagen type I mRNA were analyzed by reverse transcription-polymerase chain reaction. α-SMA protein was also detected using immunofluorescent staining. The amount of total soluble collagen was analyzed by Sircol collagen assay. ROS activity was measured by the nitroblue tetrazolium reduction assay and visualized by fluorescent microscopy. EGCG significantly inhibited expressions of α-SMA and collagen type I mRNA and reduced α-SMA and collagen protein levels at concentrations of 10-20 µg/mL. EGCG also inhibited TGF-β1-induced ROS production at the same concentrations. These results suggest the possibility that EGCG may be effective at inhibiting the development of nasal polyps through an anti-oxidant effect.

Short-Term Treatment with a SOD/Catalase Mimetic, EUK-207, Mitigates Pneumonitis and Fibrosis after Single-Dose Total-Body or Whole-Thoracic Irradiation

In the event of a radiological accident or terrorist attack, whole- or partial-body exposure can injure the lungs. To simulate such an incident, we used a single fraction of total-body irradiation (TBI) or whole-thoracic irradiation to induce pneumonitis or pulmonary fibrosis, respectively, in a rat model. The superoxide dismutase and catalase mimetic EUK-207 was given by subcutaneous injection (20 mg/kg/day, 5 days per week, once daily) starting at 7 days after irradiation and stopping before pneumonitis developed. After TBI, morbidity and the increase in breathing rates associated with pneumonitis were significantly improved in rats treated with EUK-207 compared to rats receiving irradiation alone. At 42 days after TBI (the peak of pneumonitis) changes in vascular end points including pulmonary hemodynamics ex vivo and relative arterial density in lungs were also mitigated by EUK-207. At 7 months after whole-thoracic irradiation, EUK-207 reduced synthesis of collagen as assessed by the Sircol collagen assay and Masson's trichrome staining. Our results demonstrate promise for EUK-207 as a mitigator of radiation pneumonitis and fibrosis. We also demonstrate for the first time mitigation of multiple vascular injuries in the irradiated lung in vivo by EUK-207.

10‐Hydroxy‐2‐decenoic acid prevents ultraviolet A‐induced damage and matrix metalloproteinases expression in human dermal fibroblasts

10-Hydroxy-2-decenoic acid (10-HDA) is a major fatty acid component of royal jelly, which has been reported to have a variety of beneficial pharmacological characteristics. However, the effects of 10-HDA on skin photoageing and its potential mechanism of action are unclear. We investigated the protective effects of 10-HDA on ultraviolet (UV) A-induced damage in human dermal fibroblasts (HDFs). We then explored the inhibitory effects of 10-HDA on UVA-induced matrix metalloproteinases (MMPs) expression and elucidated the signalling pathways controlling MMPs inhibition. Primary human dermal fibroblasts were exposed to UVA. Cell proliferation, cellular senescent state and collagen content were analysed using CCK-8, senescence-associated β-galactosidase staining and Sircol collagen assay, respectively. Fluorometric assays were performed to detect the formation of reactive oxygen species (ROS) in the cells. The mRNA levels of MMP-1, MMP-3 and type I (α1) collagen were determined by quantitative real-time PCR. Western blot was applied to detect the expression of MMP-1, MMP-3, JNK and p38 MAPK. HDFs treated with 10-HDA were significantly protected from UVA-induced cytotoxicity, ROS, cellular senescence and stimulated collagen production. Moreover, 10-HDA suppressed the UVA-induced expression of MMP-1 and MMP-3 at both the transcriptional and protein levels. Treatment with 10-HDA also reduced the UVA-induced activation of the JNK and p38 MAPK pathways. The data obtained in this study provide evidence that 10-HDA could prevent UVA-induced damage and inhibit MMP-1 and MMP-3 expressions. Therefore, 10-HDA may be a potential agent for the prevention and treatment of skin photoageing.

Short-Term Treatment with a SOD/Catalase Mimetic, EUK-207, Mitigates Pneumonitis and Fibrosis after Single-Dose Total-Body or Whole-Thoracic Irradiation

In the event of a radiological accident or terrorist attack, whole- or partial-body exposure can injure the lungs. To simulate such an incident, we used a single fraction of total-body irradiation (TBI) or whole-thoracic irradiation to induce pneumonitis or pulmonary fibrosis, respectively, in a rat model. The superoxide dismutase and catalase mimetic EUK-207 was given by subcutaneous injection (20 mg/kg/day, 5 days per week, once daily) starting at 7 days after irradiation and stopping before pneumonitis developed. After TBI, morbidity and the increase in breathing rates associated with pneumonitis were significantly improved in rats treated with EUK-207 compared to rats receiving irradiation alone. At 42 days after TBI (the peak of pneumonitis) changes in vascular end points including pulmonary hemodynamics ex vivo and relative arterial density in lungs were also mitigated by EUK-207. At 7 months after whole-thoracic irradiation, EUK-207 reduced synthesis of collagen as assessed by the Sircol collagen assay and Masson's trichrome staining. Our results demonstrate promise for EUK-207 as a mitigator of radiation pneumonitis and fibrosis. We also demonstrate for the first time mitigation of multiple vascular injuries in the irradiated lung in vivo by EUK-207.

Effects of PPAR-delta agonist and zinc on vaginal smooth muscle cells collagen and tropoelastin production

The objective was to measure the effects of peroxisome proliferator-activated receptor delta (PPAR-delta) agonist and zinc sulfate (ZS) on vaginal smooth muscle cell (SMC) proliferation, tropoelastin production, and collagen production. SMC cultures were performed from vaginal wall biopsies and were incubated with ZS and PPAR-delta agonist GW501516 (GW). Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Supernatants and cell lysates were collected. Tropoelastin production was measured by Fastin Elastin Assay and collagen was measured with Sircol Collagen Assay. SMC proliferation was similar with 20 μM ZS or 10 nM GW compared to control. Tropoelastin production was significantly increased by 20 μM ZS and by 10 nM GW. Cell culture surface deposited elastin production was significantly increased by 20 μM ZS and by the combination of 20 μM ZS with 10 nM GW, and collagen production was significantly increased by 10 nM GW and by the combination of 20 μM ZS with 10 nM GW. PPAR-delta agonist and ZS increases vaginal SMC tropoelastin and collagen production.

Berberine Inhibits Myofibroblast Differentiation in Nasal Polyp‐Derived Fibroblasts via the p38 Pathway

The purposes of this study were to determine whether berberine has any effect on phenotype changes and extracellular matrix (ECM) production in nasal polyp-derived fibroblasts (NPDFs) and to investigate the underlying molecular mechanism. NPDFs were pre-treated with berberine prior to induction by transforming growth factor (TGF)-β1. The expression of α-smooth muscle actin (SMA) and collagen type I mRNA was determined by a reverse transcription-polymerase chain reaction, and the expression of α-SMA protein and collagen type I was determined by western blotting and/or immunofluorescent staining. The total soluble collagen production was analysed by the SirCol collagen assay. The expression of several signaling molecules of the TGF-β1 pathway was evaluated by western blot analysis. In TGF-β1-induced NPDFs, berberine significantly inhibited the expression of α-SMA and collagen type I mRNA and reduced α-SMA and collagen protein levels. Berberine only suppressed the expression of pp38 among the evaluated signaling molecules. SB203580 (a specific inhibitor of p38 kinase) markedly suppressed the increased expression of collagen type I and α-SMA in TGF-β1-induced NPDFs. Berberine exerts suppressive effects on phenotype changes and ECM production in NPDFs via p38 signaling pathway interference. The findings provide new therapeutic options for ECM production in nasal polyps.

Paeoniae Radix Reduces PDGF-Stimulated Hepatic Stellate Cell Migration

Hepatic stellate cells (HSCs) play a key role in the pathogenesis of liver fibrosis. In chronic liver injury, HSCs undergo transdifferentiation to an activated myofibroblastic phenotype and migrate to injured areas in response to chemotactic factors, producing extracellular matrix proteins such as collagen type I to repair the damage as well as overexpression of α-smooth muscle actin (α-SMA). Paeoniae Radix, the root of Paeonia lactiflora Pall, was investigated for PDGF-BB-induced HSC chemotaxis. Rat HSCs and LX-2, a human HSC cell line, were used for the in vitro experiments. Cell migration was analyzed by wound-healing and transwell assays. An ELISA and a Sircol collagen assay kit were used to detect the expressions of α-SMA and of collagen, respectively. Phosphorylations of mitogen-activated protein kinases, including ERK 1/2, p38, and JNK, were evaluated with immunoblotting. Results indicated that PDGF-BB increased migration as well as α-SMA and collagen expression in HSCs. Paeoniae Radix extracts and its active components, paeonol and 1,2,3,4,6-penta- O-galloyl- β-D-glucose (PGG), inhibited PDGF-BB-induced HSC migration and α-SMA and collagen expressions in a concentration-dependent manner. The inhibitory effects were associated with downregulation of PDGF receptor- α, ERK, p38, and JNK activation. Both paeonol and PGG participate in HSC migration, but via differential mechanisms.

Pro-Fibrotic Role of the A2B Adenosine Receptor in Human Cardiac Fibroblasts

Background and Aims: Adenosine is a signaling nucleoside and the interstitial levels of this nucleoside are elevated in certain pathophysiological conditions. Results of our previous studies suggest that adenosine play an important role in pulmonary fibrosis. The goal of the current study is to determine the potential role of adenosine and its receptor subtypes in cardiac fibrosis using the primary human cardiac fibroblasts (HCF) as a model system. Methods: Expression of adenosine receptors (AdoR), a-smooth muscle actin and a-1 pro-collagen was determined using real-time RT-PCR. The concentrations of IL-6, soluble ST-2 and PAPPA in the cell supernatants were measured using ELISA. The concentration of soluble collagen was determined using Sircol collagen assay. Results: Among the four subtypes of AdoRs, the A2B AdoR was expressed at the highest level in HCF. NECA, a stable analog of adenosine, significantly increased the release of IL-6 in a concentration-dependent manner, with a maximal increase of 2.4 6 0.1 fold over the basal level. In addition, NECA (10mM) increased the expression of a-smooth muscle actin and a-1 pro-collagen, and the production of collagen (1.8 6 0.1 fold induction, from 3.4 6 0.2 to 6.0 6 0.4 mg/ml, p!0.05) from HCF. Furthermore, NECA increased the release of two novel biomarkers of cardiovascular diseases (CVD), soluble ST-2 (1.7 6 0.1 fold induction, from 1.5 6 0.1 to 2.6 6 0.1 ng/ml, p!0.05) and PAPPA (4.46 0.6 fold induction, from 1.46 0.5 to 6.26 0.8 ng/ml, p!0.05). The effects of NECA on release of IL-6, collagen, ST-2, and PAPPA and expression of a-smooth muscle actin and a-1 pro-collagen were completely abolished by a selective A2B AdoR antagonist, GS-6201. Conclusions: This study indicates that the A2B AdoR is the predominant subtype of AdoRs expressed in primary human HCF. Activation of this receptor increases the release of IL-6, production of collagen, expression of fibrotic markers and release of biomarkers of CVD. These findings suggest that A2B AdoR might mediate the fibrotic response in heart diseases.

Flexor Tendon Tissue Engineering: Acellularization of Human Flexor Tendons with Preservation of Biomechanical Properties and Biocompatibility

Acellular human tendons are a candidate scaffold for tissue engineering flexor tendons of the hand. This study compared acellularization methods and their compatibility with allogeneic human cells. Human flexor tendons were pretreated with 0.1% ethylenediaminetetracetic acid (EDTA) for 4 h followed by 24 h treatments of 1% Triton X-100, 1% tri(n-butyl)phosphate, or 0.1% or 1% sodium dodecyl sulfate (SDS) in 0.1% EDTA. Outcomes were assessed histologically by hematoxylin and eosin and SYTO green fluorescent nucleic acid stains and biochemically by a QIAGEN DNeasy kit, Sircol collagen assay, and 1,9 dimethylmethylene blue glycosaminoglycan assay. Mechanical data were collected using a Materials Testing System to pull to failure tendons acellularized with 0.1% SDS. Acellularized tendons were re-seeded in a suspension of human dermal fibroblasts. Attachment of viable cells to acellularized tendon was assessed biochemically by a cell viability assay and histologically by a live/dead stain. Data are reported as mean±standard deviation. Compared with the DNA content of fresh tendons (551±212 ng DNA/mg tendon), only SDS treatments significantly decreased DNA content (1% SDS [202.8±37.4 ng DNA/mg dry weight tendon]; 0.1% SDS [189±104 ng DNA/mg tendon]). These findings were confirmed by histology. There was no decrease in glycosaminoglycans or collagen following acellularization with SDS. There was no difference in the ultimate tensile stress (55.3±19.2 [fresh] vs. 51.5±6.9 [0.1% SDS] MPa). Re-seeded tendons demonstrated attachment of viable cells to the tendon surface using a viability assay and histology. Human flexor tendons were acellularized with 0.1% SDS in 0.1% EDTA for 24 h with preservation of mechanical properties. Preservation of collagen and glycoaminoglycans and re-seeding with human cells suggest that this scaffold is biocompatible. This will provide a promising scaffold for future human flexor tendon tissue engineering studies to further assess biocompatibility through cell proliferation and in vivo studies.

Changes in the Mechanical and Biochemical Properties of Aortic Tissue due to Cold Storage

Temporary cold storage is a common procedure for preserving tissues for a short time before using them in a clinical or experimental setting. The process of storing tissues at refrigeration or freezing temperatures can affect the mechanical properties of the tissue. Previous studies were limited to uniaxial tensile tests and did not have substantial explanations for what was structurally occurring in the tissue to account for the changes in mechanical properties. This study involved biaxial tensile testing of bovine thoracic aortas that had been stored at common storage temperatures (4°C, -20°C, or-80°C) for three different time points (48 h, 1 wk, or 3 wk). The slopes of the initial and stiff region of the stress-strain curves were measured. The knee point of the stress-strain curves was also determined. Collagen content before and after storage was quantified using a Sircol collagen assay kit. The stored arteries showed decreased initial slope and increased stiff slope after 48 h of 4°C refrigeration. The -20°C and -80°C storage conditions had similar initial slopes compared with the fresh ones but an increase in the stiff slope. There was also a significant shift of the knee point to a higher strain and stress. The soluble and insoluble collagen content decreased significantly due to storage but the percentage of cross-linked collagen was unchanged. Cold storage causes several changes in the mechanical properties due to structural and biochemical changes in the tissue. Overall, freezing protocols (either -20°C or -80°C) are suggested over refrigeration (4°C) for maintaining the initial stress-strain behavior.

Flutamide inhibits nifedipine- and interleukin-1β-induced collagen overproduction in gingival fibroblasts

To understand the role of the androgen receptor in gingival overgrowth, the effects of flutamide on interleukin-1 beta- and nifedipine-induced gene expression of connective tissue growth factor (CTGF/CCN2) and collagen production in gingival fibroblasts were examined. Gingival fibroblasts from healthy subjects and patients with dihydropyridine-induced gingival overgrowth (DIGO) were used. Confluent cells were treated with nifedipine, interleukin-1 beta or both. The mRNA expression was examined using real-time polymerase chain reaction, and the concentration of total soluble collagen in conditioned media was analysed by Sircol Collagen Assay. In addition, the protein expressions of androgen receptor, CTGF/CCN2 and type I collagen in gingival tissue were determined by western blot. Interleukin-1 beta was more potent than nifedipine in stimulating CTGF/CCN2 and procollagen alpha1(I) mRNA expression, and there was an additive effect of the two drugs. Healthy cells exhibited an equal or stronger response of procollagen alpha1(I) than those with DIGO, but DIGO cells displayed a stronger response in the secretion of soluble collagen in the same conditions. Flutamide, an androgen receptor antagonist, inhibited stimulation by nifedipine or interleukin-1 beta. Additionally, the protein expressions of androgen receptor and type I collagen were higher in DIGO gingival tissue than those in healthy gingival tissue. The data suggest that both nifedipine and interleukin-1 beta play an important role in DIGO via androgen receptor upregulation and that gingival overgrowth is mainly due to collagen accumulation. Flutamide decreases the gene expression and protein production of collagen from dihydropyridine-induced overgrowth cells.

Essential modification of the Sircol Collagen Assay for the accurate quantification of collagen content in complex protein solutions

Collagen contains the unique imino acid hydroxyproline (HyPro), which is involved in the stabilization of this triple helical molecule. The concentration of HyPro is customarily used to calculate the total collagen content in a cell culture environment and in acid hydrolysates of normal and pathophysiological tissues. Radiolabelling, chromatographic and calorimetric assays have been developed over the years for the accurate determination of collagen content through HyPro estimation. Recently, the Sircol Collagen Assay (SCA) has been almost exclusively adopted as the fastest and simplest colorimetric method for the determination of collagen concentration in complex protein solutions. We show here that the colorimetric SCA, which is based on the binding of Sirius red (SR) to collagen, is flawed by interference of non-collagenous proteins (e.g. serum). In fact, we demonstrate that SCA in cell culture systems and tissue hydrolysates results in a dramatic overestimation of collagen content ranging from 3- to 24-fold. In order to rescue this otherwise very practical assay, we introduce a simple purification procedure that allows the removal of interfering non-collagenous proteins from culture media and tissue samples so that accurate measurements with SCA are now possible.

Paclitaxel inhibits growth, migration and collagen production of human Tenon's fibroblasts—potential use in drug-eluting glaucoma drainage devices

Objective/AimOne of the factors limiting the long-term success of glaucoma drainage devices and traditional filtering surgery is the tendency of bleb encapsulation. Glaucoma shunts present an opportunity of introducing drug-eluting mechanisms for a lasting exposure of the bleb to anti-proliferative substances. The aim of this in vitro study was to investigate the effects of short- and long-term exposure of primary cultures of human Tenon's fibroblasts to different concentrations of paclitaxel on cell proliferation, migration, collagen production and cytotoxicity, in order to evaluate the suitability of the drug for the use in such a device.Materials/MethodsSeven individual primary cultures of human Tenon's fibroblasts were observed over the course of 1 week after administering paclitaxel concentrations varying from 10−9 mol/l to 10−6 mol/l for either 1 hour or continuously. Relative cell count and migration across a cell-free area introduced by scratching through a confluent cell layer were determined every 24 hours, using photomicrographs of the cells for each concentration and exposure time. Soluble collagen concentration in the cell culture medium was determined using a Sircol collagen assay 72 hours after paclitaxel exposure. Cytotoxicity of the compound was assessed by flow cytometry using dual staining with annexin V-FITC and propidium iodide.ResultsPaclitaxel dose-dependently inhibited both proliferation and migration of the cells. Cell count was reduced at all concentrations and both exposure times (p = 0.001); similarly, all but two concentrations of paclitaxel caused a significant reduction of cell migration (p < 0.001). This may be explained in part by the dose- and time-dependent induction of apoptosis in up to 23.7% of the cells (maximal effect at 10−6 mol/l, 7 days after exposure). Collagen production was significantly reduced at all concentrations and at both exposure times. However, no statistically significant difference was observed between any of the concentrations, indicating that this inhibition may be an indirect effect.ConclusionPaclitaxel may be a useful addition to the repertoire of anti-proliferative substances currently in use in glaucoma filtering surgery and shunt implantation. Further studies of the compound and its effects on Tenon's fibroblasts as well as other ocular tissues are warranted.

Histone deacetylase 7, a potential target for the antifibrotic treatment of systemic sclerosis

We have recently shown a significant reduction in cytokine-induced transcription of type I collagen and fibronectin in systemic sclerosis (SSc) skin fibroblasts upon treatment with trichostatin A (TSA). Moreover, in a mouse model of fibrosis, TSA prevented the dermal accumulation of extracellular matrix. The purpose of this study was to analyze the silencing of histone deacetylase 7 (HDAC-7) as a possible mechanism by which TSA exerts its antifibrotic function. Skin fibroblasts from patients with SSc were treated with TSA and/or transforming growth factor beta. Expression of HDACs 1-11, extracellular matrix proteins, connective tissue growth factor (CTGF), and intercellular adhesion molecule 1 (ICAM-1) was analyzed by real-time polymerase chain reaction, Western blotting, and the Sircol collagen assay. HDAC-7 was silenced using small interfering RNA. SSc fibroblasts did not show a specific pattern of expression of HDACs. TSA significantly inhibited the expression of HDAC-7, whereas HDAC-3 was up-regulated. Silencing of HDAC-7 decreased the constitutive and cytokine-induced production of type I and type III collagen, but not fibronectin, as TSA had done. Most interestingly, TSA induced the expression of CTGF and ICAM-1, while silencing of HDAC-7 had no effect on their expression. Silencing of HDAC-7 appears to be not only as effective as TSA, but also a more specific target for the treatment of SSc, because it does not up-regulate the expression of profibrotic molecules such as ICAM-1 and CTGF. This observation may lead to the development of more specific and less toxic targeted therapies for SSc.

Interleukin-4 induces the activation and collagen production of cultured human intrahepatic fibroblasts via the STAT-6 pathway

Interleukin-4 (IL-4) is overexpressed in liver grafts in a context of severe recurrent hepatitis C, during which the development of fibrosis is dramatically accelerated. In this study, we examined the effects of IL-4 on the activation and collagen production of cultured human intrahepatic (myo)fibroblasts (hIHFs), and investigated the underlying mechanisms. The myofibroblastic nature of cells was evaluated morphologically using activation markers (smooth muscle α-actin, vimentin and prolyl 4-hydroxylase). Quiescent hIHFs were obtained by cell incubation in serum-free medium or cell culture on Matrigel. We first analyzed IL-4 receptor expression, STAT-6 activation by IL-4, and STAT-6 inhibition by an anti-IL-4 antibody or by STAT-6 small-interfering RNA (siRNA) transfection. We then focused on collagen production, using quantitative real-time PCR to analyze the effect of IL-4 on the mRNA expression of collagens I, III and IV, and on collagen levels in supernatants of hIHFs, using the Sircol collagen assay. hIHFs cultured in plastic wells appeared to be morphologically activated. The expression of activation markers was reduced by serum deprivation or culture on Matrigel, and restored by IL-4 incubation. The IL-4 receptor was expressed by hIHFs, and STAT-6 was activated following incubation with IL-4. Both anti-IL-4 antibody and STAT-6 siRNA transfection inhibited this activation. The treatment of hIHFs with IL-4 increased the mRNA expression of collagens I, III and IV (P<0.05) and elevated collagen levels in supernatants (P=0.01 vs untreated cells). Therefore, IL-4 exerts profibrotic effects by activating hIHFs and inducing collagen production and secretion. This effect requires IL4-R binding and STAT-6 activation. IL-4 may thus be involved in accelerated course of fibrogenesis during recurrent hepatitis C.

Increased renal collagen cross‐linking and lipid accumulation in nephropathy of Zucker diabetic fatty rats

Zucker diabetic fatty (ZDF) rat is a genetic model of type 2 diabetes and obesity. The mechanism underlying nephropathy in ZDF rats, however, remains unclear. ZDF rats were compared to age-matched Zucker lean (ZL) rats. Physiological and blood biochemical parameters, renal glomerular cross-sectional area (hematoxylin-eosin staining), fibrosis (van Giesen staining), collagen composition (Sircol Collagen Assay), lipids (enzymatic method) and mRNA expression (RT-PCR) were determined. ZDF rats showed an increase in renal-insoluble collagen content and the ratio of renal-insoluble to salt-soluble collagen (2- and 1.5-fold of the control animals). There were increases in renal glomerulosclerosis and interstitial fibrosis in ZDF rats (increased to 2-fold) in the glomerular mesangium and tubulointerstitium, and increased glomerular area. Renal triglyceride accumulated to greater than 2-fold of those levels in ZL rats. These changes were accompanied by hypoalbuminemia, and elevated plasma blood urea nitrogen and uric acid levels. Gene profiling showed increased expression of transcripts encoding the glomerulosclerotic mediator collagens I and IV, plasminogen activator inhibitor-1, transforming growth factor-beta1, and angiotensin II type 1 receptor in ZDF rat kidney. Moreover, renal expression of mRNAs encoding sterol regulatory element-binding protein-1, a nuclear transcription factor that activates genes involved in fatty acid synthesis, and acetyl-CoA carboxylase, a key enzyme that mediates fatty acid synthesis, was increased in ZDF rats. Our findings suggest that dysregulated gene expression may result in increased renal collagen cross-linking and lipid accumulation, that may be associated with development of nephropathy in the animal model of type 2 diabetes and obesity.

Src kinases in systemic sclerosis: Central roles in fibroblast activation and in skin fibrosis

Src kinases are nonreceptor tyrosine kinases, which have been implicated in cytoskeletal organization and cell mobility. This study was undertaken to evaluate the potential of Src kinases as novel targets of antifibrotic therapies. Fibroblast cultures were obtained from 10 patients with systemic sclerosis (SSc) and 5 healthy subjects. Src signaling was inhibited using small-molecule inhibitors and overexpression of a dominant-negative mutant of Src and of the endogenous inhibitor Csk. The expression of extracellular matrix proteins was analyzed by real-time polymerase chain reaction and by SirCol collagen assay. Toxic effects were excluded by MTT assay and staining for annexin V and propidium iodide. The mouse model of bleomycin-induced dermal fibrosis was used to assess the role of Src kinases in dermal fibrosis in vivo. Stimulation with transforming growth factor beta and platelet-derived growth factor activated Src signaling in dermal fibroblasts from patients with SSc and healthy donors. Incubation with the Src kinase inhibitors or overexpressed mutant Src or Csk reduced the synthesis of messenger RNA for COL1A1, COL1A2, and fibronectin 1. A dose-dependent reduction in collagen release was also observed at the protein level. No inhibitory effects on proliferation and no increase in the number of apoptotic or necrotic fibroblasts were observed. Consistent with the in vitro data, inhibition of Src kinases prevented experimental dermal fibrosis. Dermal thickness, the amount of collagen protein, and the number of myofibroblasts were reduced in a dose-dependent manner. These findings indicate that Src kinases play important roles in the activation of fibroblasts and in the development of experimental fibrosis. Thus, Src kinases might be interesting targets for novel antifibrotic therapies in SSc.

W1830 Polyphenon E, An EGCG Containing Green Tea Extract That Inhibits Tumor Growth, Has No Impact On Wound Collagen Content or Clinical Rate of Wound Infection or Dehiscence Yet Has a Mild Inhibitory Effect On Healing As Judged By Tensometry

Introduction: Catechins in green tea, in particular EGCG (epigallocatechin-3-gallate), inhibit tumor growth in many murine models. Polyphenon E (PolyE) is a concentrated mixture of catechins (65% EGCG) with minimal side affects in humans that has stronger anti-cancer effects than tea alone. Surgery is associated with increased rates of tumor growth and metastases postoperatively(postop) in the murine setting. Perioperative (periop) administration of Poly E may limit surgery's tumor stimulatory effects. Prior to a Phase 1 study, PolyE's effects on wound healing must be studied. This study's purpose was to assess PolyE's impact on laparotomywound strength and collagen content. Methods: Balb/Cmice were randomized to plain water (Control, n=20) or a 1% PolyE solution (n=25) for drinking;10 days later all mice underwent sham laparotomy that was suture closed. Mice were sacrificed on postop days (PODs) 7 or 21 and the abdominal wall pelts were harvested. The peak force and total energy required to rupture each wound was determined via tensometry; wound collagen content was determined via a Sircol Collagen assay. Serum EGCG levels were determined in 3 PolyE mice on POD1, 7, and 21 to verify drug ingestion. Results are presented as mean ± SD; data was assessed using the student's t-test and Mann-Whitney U-test (significance, p<0.05). Results: No wound infections or dehiscences were noted in either group. The mean peak rupture force for each group was statistically similar on POD 7 and 21. There was no statistical difference in the total energy required for rupture on POD 7, however, on POD 21 the total energy required in the PolyE group (4.1 ± 0.78 N-mm) was significantly lower than for the control mice (5.6 ± 1.7 N-mm; p=0.026). When the 2 groups wound collagen results on POD 7 were compared there was no difference noted, the same was true for the POD 21 results. EGCG was detected in the PolyE mice serum on POD 1 and POD 7; on POD21 very low levels were detected. Conclusions: Perioperative PolyE administration was not associated with wound complications. The peak force for rupture and the collagen content was similar at both time points for the 2 groups as was total energy for rupture on POD 7; total energy on POD 21 was less for the PolyE group. PolyE warrants further study as a perioperative anti-cancer agent in the cancer setting.

All-trans retinoic acid regulates collagen synthesis in human uterine leiomyomas

All-trans retinoic acid regulates collagen synthesis in human uterine leiomyomas

Trichostatin A prevents the accumulation of extracellular matrix in a mouse model of bleomycin‐induced skin fibrosis

Tissue fibrosis is a hallmark compromising feature of many disorders. In this study, we investigated the antifibrogenic effects of the histone deacetylase inhibitor trichostatin A (TSA) on cytokine-driven fibrotic responses in vitro and in vivo. Skin fibroblasts from patients with systemic sclerosis (SSc) and normal healthy control subjects were stimulated with profibrotic cytokines in combination with TSA. Human Colalpha1(I) and fibronectin were measured using real-time polymerase chain reaction, and levels of soluble collagen were estimated using the SirCol collagen assay. Electromobility shift assay and confocal fluorescence microscopy were used to investigate the intracellular distribution of Smad transcription factors. For in vivo analysis, skin fibrosis was quantified by histologic assessment of mouse skin tissue in a model of bleomycin-induced fibrosis. Reductions in the cytokine-induced transcription of Colalpha1(I) and fibronectin were observed in both normal and SSc skin fibroblasts following the addition of TSA. Similarly, the expression of total collagen protein in TSA-stimulated SSc skin fibroblasts was reduced to basal levels. The mechanism of action of TSA included inhibition of the nuclear translocation and DNA binding of profibrotic Smad transcription factors. Western blot analysis revealed an up-regulation of the cell cycle inhibitor p21 by TSA, leading to reduced proliferation of fibroblasts. In addition, in bleomycin-induced fibrosis in mice, TSA prevented dermal accumulation of extracellular matrix in vivo. These findings provide novel insights into the epigenetic regulation of fibrosis. TSA and similar inhibitory compounds appear to represent early therapeutic strategies for achieving reversal of the cytokine-driven induction of matrix synthesis that leads to fibrosis.