Biological effects of a new ultraviolet A1 prototype based on light-emitting diodes on the treatment of localized scleroderma.

Ultraviolet A1 (UVA1 ) phototherapy (spectral range 340-400 nm) is a well-established treatment option for various skin diseases such as localized scleroderma. Recent improvements of conventional UVA1 light sources (metal-halide or fluorescent lamps) have brought attention to a new light-emitting diode (LED) technology with remarkable advantages in handling and clinical routine. This study provides a preclinical histological and molecular evaluation of an LED-based UVA1 prototype with a narrower spectral range (360-400 nm) for treating localized scleroderma. Scleroderma mouse models and fibroblasts in vitro were exposed to LED-based UVA1 phototherapy or to irradiation with a commercially available metal-halide lamp emitting low-dose (20, 40 J/cm2 ), medium-dose (60 J/cm2 ) and high-dose (80, 100 J/cm2 ) UVA1 light. Both UVA1 light sources affected inflammatory genes (IL-1α and IL-6) and growth factors (TGFß-1 and TGFß-2). Increased collagen type 1 was reduced after UVA1 phototherapy. Matrix metalloproteinase-1 was more enhanced after a medium dose of LED-based UVA1 phototherapy than after conventional treatment. In vivo, dermal thickness and the amount of collagen were reduced after both treatment methods. Remarkably, myofibroblasts were more effectively reduced by a medium dose of LED-based UVA1 phototherapy. The study indicates that LED-based UVA1 phototherapy yields similar or even better results than conventional treatment. In terms of biosafety and patient comfort, LED-based UVA1 phototherapy offers clear advantages over conventional treatment because of the use of a narrower and less harmful UVA1 spectrum, less heat generation and shorter treatment times at the same irradiation intensity. Clinical studies are required to confirm these results in patients with localized scleroderma.

Effect of tamoxifen on fibrosis, collagen content and transforming growth factor-ß1, -ß2 and -ß3 expression in common bile duct anastomosis of pigs.

End-to-end anastomosis in the treatment for bile duct injury during laparoscopic cholecystectomy has been associated with stricture formation. The aim of this study was to experimentally investigate the effect of oral tamoxifen (tmx) treatment on fibrosis, collagen content and transforming growth factor-ß1, -ß2 and -ß3 expression in common bile duct anastomosis of pigs. Twenty-six pigs were divided into three groups [sham (n = 8), control (n = 9) and tmx (n = 9)]. The common bile ducts were transected and anastomosed in the control and tmx groups. Tmx (40 mg/day) was administered orally to the tmx group, and the animals were euthanized after 60 days. Fibrosis was analysed by Masson's trichrome staining. Picrosirius red was used to quantify the total collagen content and collagen type I/III ratio. mRNA expression of transforming growth factor (TGF)-ß1, -ß2 and -ß3 was quantified using real-time polymerase chain reaction (qRT-PCR). The control and study groups exhibited higher fibrosis than the sham group, and the study group showed lower fibrosis than the control group (P = 0.011). The control and tmx groups had higher total collagen content than the sham group (P = 0.003). The collagen type I/III ratio was higher in the control group than in the sham and tmx groups (P = 0.015). There were no significant differences in the mRNA expression of TGF-ß1, -ß2 and -ß3 among the groups (P > 0.05). Tmx decreased fibrosis and prevented the change in collagen type I/III ratio caused by the procedure.

Transcriptomic-metabolomic reprogramming in EGFR-mutant NSCLC early adaptive drug escape linking TGFß2-bioenergetics-mitochondrial priming.

The impact of EGFR-mutant NSCLC precision therapy is limited by acquired resistance despite initial excellent response. Classic studies of EGFR-mutant clinical resistance to precision therapy were based on tumor rebiopsies late during clinical tumor progression on therapy. Here, we characterized a novel non-mutational early adaptive drug-escape in EGFR-mutant lung tumor cells only days after therapy initiation, that is MET-independent. The drug-escape cell states were analyzed by integrated transcriptomic and metabolomics profiling uncovering a central role for autocrine TGFß2 in mediating cellular plasticity through profound cellular adaptive Omics reprogramming, with common mechanistic link to prosurvival mitochondrial priming. Cells undergoing early adaptive drug escape are in proliferative-metabolic quiescent, with enhanced EMT-ness and stem cell signaling, exhibiting global bioenergetics suppression including reverse Warburg, and are susceptible to glutamine deprivation and TGFß2 inhibition. Our study further supports a preemptive therapeutic targeting of bioenergetics and mitochondrial priming to impact early drug-escape emergence using EGFR precision inhibitor combined with broad BH3-mimetic to interrupt BCL-2/BCL-xL together, but not BCL-2 alone.

Structural and molecular features of intestinal strictures in rats with Crohn's-like disease.

AIM: To develop a new rat model we wanted to gain a better understanding of stricture formation in Crohn's disease (CD). METHODS: Chronic colitis was induced locally by the administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS). The relapsing inflammation characteristic to CD was mimicked by repeated TNBS treatments. Animals were randomly divided into control, once, twice and three times TNBS-treated groups. Control animals received an enema of saline. Tissue samples were taken from the strictured colonic segments and also adjacent proximally and distally to its 60, 90 or 120 d after the last TNBS or saline administrations. The frequency and macroscopic extent of the strictures were measured on digital photographs. The structural features of strictured gut wall were studied by light- and electron microscopy. Inflammation related alterations in TGF-beta 2 and 3, matrix metalloproteinases 9 (MMP9) and TIMP1 mRNA and protein expression were determined by quantitative real-time PCR and western blot analysis. The quantitative distribution of caspase 9 was determined by post-embedding immunohistochemistry. RESULTS: Intestinal strictures first appeared 60 d after TNBS treatments and the frequency of them increased up to day 120. From day 90 an intact lamina epithelialis, reversible thickening of lamina muscularis mucosae and irreversible thickening of the muscularis externa were demonstrated in the strictured colonic segments. Nevertheless the morphological signs of apoptosis were frequently seen and excess extracellular matrix deposition was recorded between smooth muscle cells (SMCs). Enhanced caspase 9 expression on day 90 in the SMCs and on day 120 also in myenteric neurons indicated the induction of apoptosis. The mRNA expression profile of TGF-betas after repeated TNBS doses was characteristic to CD, TGF-beta 2, but not TGF-beta 3 was up-regulated. Overexpression of MMP9 and down-regulation of TIMP1 were demonstrated. The progressive increase in the amount of MMP9 protein in the strictures was also obvious between days 90 and 120 but TIMP1 protein was practically undetectable at this time. CONCLUSION: These findings indicate that aligned structural and molecular changes in the gut wall rather than neuronal cell death play the primary role in stricture formation.

TGFß2 regulates hypothalamic Trh expression through the TGFß inducible early gene-1 (TIEG1) during fetal development.

The hypothalamus regulates the homeostasis of the organism by controlling hormone secretion from the pituitary. The molecular mechanisms that regulate the differentiation of the hypothalamic thyrotropin-releasing hormone (TRH) phenotype are poorly understood. We have previously shown that Klf10 or TGFß inducible early gene-1 (TIEG1) is enriched in fetal hypothalamic TRH neurons. Here, we show that expression of TGFß isoforms (1-3) and both TGFß receptors (TßRI and II) occurs in the hypothalamus concomitantly with the establishment of TRH neurons during late embryonic development. TGFß2 induces Trh expression via a TIEG1 dependent mechanism. TIEG1 regulates Trh expression through an evolutionary conserved GC rich sequence on the Trh promoter. Finally, in mice deficient in TIEG1, Trh expression is lower than in wild type animals at embryonic day 17. These results indicate that TGFß signaling, through the upregulation of TIEG1, plays an important role in the establishment of Trh expression in the embryonic hypothalamus.

Mycophenolate antagonizes IFN-γ-induced catagen-like changes via ß-catenin activation in human dermal papilla cells and hair follicles.

Recently, various immunosuppressant drugs have been shown to induce hair growth in normal hair as well as in alopecia areata and androgenic alopecia; however, the responsible mechanism has not yet been fully elucidated. In this study, we investigate the influence of mycophenolate (MPA), an immunosuppressant, on the proliferation of human dermal papilla cells (hDPCs) and on the growth of human hair follicles following catagen induction with interferon (IFN)-γ. IFN-γ was found to reduce ß-catenin, an activator of hair follicle growth, and activate glycogen synthase kinase (GSK)-3ß, and enhance expression of the Wnt inhibitor DKK-1 and catagen inducer transforming growth factor (TGF)-ß2. IFN-γ inhibited expression of ALP and other dermal papillar cells (DPCs) markers such as Axin2, IGF-1, and FGF 7 and 10. MPA increased ß-catenin in IFN-γ-treated hDPCs leading to its nuclear accumulation via inhibition of GSK3ß and reduction of DKK-1. Furthermore, MPA significantly increased expression of ALP and other DPC marker genes but inhibited expression of TGF-ß2. Therefore, we demonstrate for the first time that IFN-γ induces catagen-like changes in hDPCs and in hair follicles via inhibition of Wnt/ß-catenin signaling, and that MPA stabilizes ß-catenin by inhibiting GSK3ß leading to increased ß-catenin target gene and DP signature gene expression, which may, in part, counteract IFN-γ-induced catagen in hDPCs.

Genomic biomarkers for cardiotoxicity in rats as a sensitive tool in preclinical studies.

The development of safer drugs is a high priority for pharmaceutical companies. Among the various toxicities caused by drugs, cardiotoxicity is an important issue because of its lethality. In addition, cardiovascular toxicity leads to the attrition of many drug candidates in both preclinical and clinical phases. Although histopathological and blood chemistry examinations are the current gold standards for detecting cardiotoxicity in preclinical studies, the large number of withdrawals from clinical studies owing to safety problems indicate that a more sensitive tool is required. We recently identified 32 genes that were candidate genomic biomarkers for cardiotoxicity in rats. Based on their functions, the present study focused on 8 of these 32 genes (Spp1, Fhl1, Timp1, Serpine1, Bcat1, Lmcd1, Rnd1 and Tgfb2). Diagnostic accuracy for the genes was determined by a receiver-operating characteristic (ROC) analysis using more cardiotoxic and non-cardiotoxic compounds. In addition, an optimized support vector machine (SVM) model that was composed of Spp1 and Timp1 was newly constructed. This new multi-gene model exhibited a much higher diagnostic accuracy than that observed for plasma cardiac troponin I (cTnI), which is one of the most useful plasma biomarkers for cardiotoxicity detection. Furthermore, we determined that this multi-gene model could predict potential cardiotoxicity in rats in the absence of any cardiac histopathological lesions or elevations of plasma cTnI. Overall, this multi-gene model exhibited advantages over classic tools commonly used for cardiotoxicity evaluations in rats. Our current results suggest that application of the model could potentially lead to the production of safer drugs.

Dietary transforming growth factor-beta 2 (TGF-ß2) supplementation reduces methotrexate-induced intestinal mucosal injury in a rat.

BACKGROUND/AIMS: Dietary supplementation with transforming growth factor-beta (TGF-ß) has been proven to minimize intestinal damage and facilitate regeneration after mucosal injury. In the present study, we evaluated the effects of oral TGF-ß2 supplementation on intestinal structural changes, enterocyte proliferation and apoptosis following methotrexate (MTX)-induced intestinal damage in a rat and in a cell culture model. METHODS: Caco-2 cells were treated with MTX and were incubated with increasing concentrations of TGF-ß2. Cell apoptosis was assessed using FACS analysis by annexin staining and cell viability was monitored using Trypan Blue assay. Male rats were divided into four experimental groups: Control rats, CONTR- TGF-ß rats were treated with diet enriched with TGF-ß2, MTX rats were treated with a single dose of methotrexate, and MTX- TGF-ß rats were treated with diet enriched with TGF-ß2. Intestinal mucosal damage, mucosal structural changes, enterocyte proliferation and enterocyte apoptosis were determined at sacrifice. Real Time PCR and Western blot were used to determine bax and bcl-2 mRNA, p-ERK, ß-catenin, IL-1B and bax protein expression. RESULTS: Treatment of MTX-pretreated Caco-2 cells with TGF-B2 resulted in increased cell viability and decreased cell apoptosis. Treatment of MTX-rats with TGF-ß2 resulted in a significant increase in bowel and mucosal weight, DNA and protein content, villus-height (ileum), crypt-depth (jejunum), decreased intestinal-injury score, decreased level of apoptosis and increased cell proliferation in jejunum and ileum compared to the untreated MTX group. MTX-TGF-ß2 rats demonstrated a lower bax mRNA and protein levels as well as increased bcl-2 mRNA levels in jejunum and ileum compared to MTX group. Treatment with TGF-ß2 also led to increased pERK, IL-1B and ß-catenin protein levels in intestinal mucosa. CONCLUSIONS: Treatment with TGF-ß2 prevents mucosal-injury, enhances p-ERK and ß-catenin induced enterocyte proliferation, inhibits enterocyte apoptosis and improves intestinal recovery following MTX-induced intestinal-mucositis in rats.

Intratracheal bleomycin causes airway remodeling and airflow obstruction in mice.

In addition to parenchymal fibrosis, fibrotic remodeling of the distal airways has been reported in interstitial lung diseases. Mechanisms of airway wall remodeling, which occurs in a variety of chronic lung diseases, are not well defined and current animal models are limited. The authors quantified airway remodeling in lung sections from subjects with idiopathic pulmonary fibrosis (IPF) and controls. To investigate intratracheal bleomycin as a potential animal model for fibrotic airway remodeling, the authors evaluated lungs from C57BL/6 mice after bleomycin treatment by histologic scoring for fibrosis and peribronchial inflammation, morphometric evaluation of subepithelial connective tissue volume density, TUNEL (terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling) assay, and immunohistochemistry for transforming growth factor ß1 (TGFß1), TGFß2, and the fibroblast marker S100A4. Lung mechanics were determined at 3 weeks post bleomycin. IPF lungs had small airway remodeling with increased bronchial wall thickness compared to controls. Similarly, bleomycin-treated mice developed dose-dependent airway wall inflammation and fibrosis and greater airflow resistance after high-dose bleomycin. Increased TUNEL(+) bronchial epithelial cells and peribronchial inflammation were noted by 1 week, and expression of TGFß1 and TGFß2 and accumulation of S100A4(+) fibroblasts correlated with airway remodeling in a bleomycin dose-dependent fashion. IPF is characterized by small airway remodeling in addition to parenchymal fibrosis, a pattern also seen with intratracheal bleomycin. Bronchial remodeling from intratracheal bleomycin follows a cascade of events including epithelial cell injury, airway inflammation, profibrotic cytokine expression, fibroblast accumulation, and peribronchial fibrosis. Thus, this model can be utilized to investigate mechanisms of airway remodeling.

Inhibition effect of tetrandrine on haze formation after Epi-LASIK surgery in rabbits.

PURPOSE: To observe the inhibitory effect of tetrandrine on haze formation after Epi-LASIK surgery in rabbits. MATERIALS AND METHODS: Twenty-seven healthy New Zealand rabbits were selected and underwent Epi-LASIK surgery. According to a self comparison principle, these rabbits were randomly divided into three groups. The first group was treated with tetrandrine eye drops (Tet), the second (negative control, NC) group was treated with pure solvent, and the third group was treated with fluorometholone (FML) solvent. Haze grades of each group were respectively observed by slit lamp exam at half-month, one month, and two months after surgery. After corneal tissues were extracted, optical microscopy, Sirius Red staining, immunohistochemistry, and semi-quantitative RT-PCR tests were conducted, in order to test collagen formation of operated eyes after surgery and expression of transformation growth factor beta 2 (TGF-ß(2)) in corneal stroma. RESULTS: At a half month and one month after surgery, haze grades and type III collagen expression in Tet and FML groups were significantly lower than that in NC groups (P < 0.01). No statistical difference was observed between Tet and FML groups. Immunohistochemistry showed that, at each time point after surgery, expressions of TGF-ß(2) protein in Tet and FML groups were significantly lower than that in the NC group (P < 0.01), whereas there was no statistical difference between Tet and FML groups. The expression level of TGF-ß(2) protein increased, reaching its peak one month after surgery. RT-PCR also showed that, at each point after surgery, the TGF-ß(2) mRNA expression in Tet and FML groups was lower than that in the NC group (P < 0.01), nevertheless no statistical difference was observed between Tet and FML groups. CONCLUSIONS: Like FML, Tet could inhibit haze formation in rabbits after Epi-LASIK surgery, possible through TGF-ß(2)-collagen-III pathway.

Relationships of a transforming growth factor-beta2 single nucleotide polymorphism and messenger ribonucleic acid abundance with bone and production traits in chickens.

Osteoporosis is a serious problem for the laying hen industry with economic, production, and welfare consequences. Transforming growth factor-beta2 (TGFbeta2) has been implicated as an important factor in coupling bone resorption and formation in bone remodeling. The current study was designed to determine if TGFbeta2 was associated with variation in bone mineralization in chickens, using 2 complementary experimental approaches. First, an intronic single nucleotide polymorphism (SNP) present in TGFbeta2 was investigated in an F(2) population to determine its association with bone, growth, and egg traits of importance to the layer and broiler industries. The TGFbeta2 SNP was significantly associated (P < 0.05) with bone mineral density and content. However, these associations became nonsignificant when BW was included as a covariate in analyses. The TGFbeta2 SNP was also significantly associated (P < 0.05) with BW from 1 to 6 wk of age and egg production from 46 to 55 wk of age. To further explore the relationship between TGFbeta2 and bone strength, bone marrow TGFbeta2 mRNA abundance was compared between broiler and layer chickens at 15, 35, and 60 wk of age. Bone and egg traits were measured along with mRNA abundance at each age and found to differ significantly between lines. The TGFbeta2 mRNA abundance was approximately 4-fold greater in broiler compared with layer hens at 15 wk of age but was similar between lines at later ages. Thus, even though the TGFbeta2 SNP will likely not be an effective marker for improving bone strength independently of changes in BW, further research is warranted to investigate the relationship of TGFbeta2 mRNA abundance to bone strength in laying hens.