Theacrine alleviates chronic inflammation by enhancing TGF-ß-mediated shifts via TGF-ß/SMAD pathway in Freund's incomplete adjuvant-induced rats.

Rheumatoid arthritis is a chronic and systemic autoimmune disease, which affects approximately 1% of the adult population worldwide. The present study investigated the therapeutic effect of theacrine (TC) on arthritis and its mechanisms in Freund's incomplete adjuvant (FIA)-induced SD rats. Rats were randomly divided into 5 groups: i) healthy control; ii) model; iii) positive control with methotrexate (MTX); iv) treatment with 12.5 mg/kg TC; and v) treatment with 25.0 mg/kg TC. The apparent scores, including changes in body weights, degree of paw swelling and arthritis indicators, were analyzed to evaluate the anti-chronic inflammatory effect of TC. The levels of interleukin (IL)-6 and transforming growth factor-ß (TGF-ß) in serum were measured by enzyme-linked immunosorbent assay. The protein and RNA expression levels of the critical factors in rats were measured to elucidate the mechanisms responsible for chronic inflammation and to verify molecular indexes of chronic inflammatory conditions. TC notably suppressed the severity of FIA-induced rat by attenuating the apparent scores, animal weight and inflammatory indexes in the 25 mg/kg TC group compared with the FIA rat model. Furthermore, TC significantly decreased the levels of IL-6 and increased the levels of TGF-ß. Histopathological examinations indicated that TC rescued the synovial hyperplasia and inflammatory cell infiltration in joint tissues. In addition, TC enhanced TGF-ß-mediated shifts in inflammatory marker expression in joint tissue. Overall, the present study demonstrated that TC exerted a superior anti-arthritic effect via the suppression of IL-6 and the activation of TGF-ß by the TGF-ß/SMAD pathway.

Effect of astragaloside IV on cognitive dysfunction in rats with cerebrally infarcted via TGF-ß / Smad signaling pathway.

Cerebral infarction is an acute cerebrovascular disease caused by abnormal blood circulation in the brain. In the present study, we investigate the effect of astragaloside IV on cognitive dysfunction in cerebrally infarcted rats via transforming growth factor-ß (TGF-ß) / Smad signaling pathway. For this purpose, 45 rats were divided into three groups including astragaloside, model, and control. 30 of 45 healthy adult male SD rats were randomly selected to establish an acute cerebral infarction model. 15 modeled rats were enrolled as a model and astragaloside group, and another 15 rats as a blank control group. The rats in the astragaloside group were fed with astragaloside IV according to 1.08 g/kg body weight, and those in the blank group and model group were given matching normal saline. The levels of TGF-ß, Smad1, Smad3 and Smad7 of TGF-ß/Smad signaling transduction pathway at T0 (week 0), T1 (week 3) and T2 (week 6) were determined by enzyme-linked immunosorbent assay (ELISA). The modified neurological severity score (mNSS) was used to evaluate the improvement of cognitive dysfunction in rats. The mNSS of rats with cerebral infarction in the astragaloside group was lower than that in the control group and model group (P< 0.05). While the levels of TGF-ß, Smad1, Smad3 and Smad7 in the astragaloside group were higher than those in the control group and model group (P< 0.05). Astragaloside IV plays an important role in improving cognitive dysfunction in rats with cerebral infarction while affecting the levels of TGF-ß, Smad1, Smad3 and Smad7 and activating TGF-ß / Smad signaling pathway.

Herbal compound 861 prevents hepatic fibrosis by inhibiting the TGF-ß1/Smad/SnoN pathway in bile duct-ligated rats.

BACKGROUND: This study was to evaluate the effects of herbal compound 861 (Cpd861) on ski-related novel protein N (SnoN) and transforming growth factor-ß1 (TGF-ß1) /Smad signaling in rats with bile duct ligation (BDL)-induced hepatic fibrosis, and to explore the mechanisms of Cpd861 on hepatic fibrosis. METHODS: Thirty Wistar male rats were randomly divided into three groups: sham operation, BDL, and Cpd861. To induce hepatic fibrosis, BDL and Cpd861 group rats underwent bile duct ligation. Cpd861 at 9 g/kg/d or an equal volume of normal saline was administered intragastrically for 28 days. Liver injury was assessed biochemically and histologically. Protein and mRNA changes for SnoN and TGF-ß1/Smad signaling (TGF-ß1, Smad2, phosphorylated Smad2 [p-Smad2], phosphorylated Smad3 [p-Smad3], fibronectin, and collagen III) were determined by Western blotting and quantitative real-time PCR. RESULTS: BDL rats treated with Cpd861 had significantly alleviated hepatic fibrosis compared to BDL rats not receiving Cpd861 treatment. Moreover, Cpd861 decreased the expression of fibrosis-associated proteins fibronectin and collagen III in liver tissue. Cpd861 administration increased the expression of SnoN protein, did not change SnoN mRNA level, and decreased TGF-ß1, p-Smad2, and p-Smad3 protein expression compared to BDL without Cpd861 treatment. CONCLUSIONS: Cpd861 attenuates hepatic fibrosis by increasing SnoN protein expression and inhibiting the TGF-ß1/Smad signaling pathway.

Alteration of TGF-ß-ALK-Smad signaling in hyperoxia-induced bronchopulmonary dysplasia model of newborn rats.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is a main chronic lung disease commonly occurs in preterm infants. BPD is characterized by impaired alveolarization and vascularization of the developing lung. Transforming growth factor-ß (TGF-ß) signaling pathway is known to play an important role during lung vascular development. In the present study, we examined whether the regulation of TGF-ß-ALK-Smad signaling pathway influence on the disruption of pulmonary vascular development in newborn rats as hyperoxia-induced BPD model. MATERIALS AND METHODS: Newborn rats were continuously exposed to 21% or 85% O2 for 7 days, and subsequently kept in normoxic condition for another 14 days. Lung tissues harvested at each time point were evaluated for the expression of TGF-ß1, ALK1, ALK5, phosphorylated Smad1/5, phosphorylated Smad2/3, VEGF, and endoglin, as accessed by both biochemical and immunohistological analyses. RESULTS: Double-fluorescence immunohistochemical staining indicated these molecules were mainly expressed in pulmonary endothelial cells. The expression of TGF-ß1 and ALK5 mRNA and protein were significantly increased in D5 hyperoxia group, while that of ALK1 mRNA and protein were significantly decreased. The level of phosphorylated Smad1/5 was significantly decreased in D7 hyperoxia group, whereas that of phosphorylated Smad2/3 was oppositely increased. In addition, the expression of vascular endothelial growth factor (VEGF) mRNA was increased at D1 with subsequent decrease in D7 hyperoxia group. There was no significantly difference in endoglin expression in entire experimental period. CONCLUSION: These results indicate that exposure to hyperoxia altered the balance between TGF-ß-ALK1-Smad1/5 and TGF-ß-ALK5-Smad2/3 pathways in pulmonary endothelial cells, which may ultimately lead to the development of BPD.

Oxymatrine inhibits aldosterone-induced rat cardiac fibroblast proliferation and differentiation by attenuating smad-2,-3 and-4 expression: an in vitro study.

BACKGROUND: We previously demonstrated oxymatrine, an alkaloid from the Chinese medicine radix Sophorae flavescentis, ameliorates hemodynamic disturbances and cardiac fibrosis; however, the underlying mechanisms are unclear. Here, we investigated the effect and mechanism of action of oxymatrine on aldosterone-induced cardiac fibroblast to myofibroblast differentiation in vitro. METHODS: Cardiac fibroblasts were isolated purified from neonatal Sprague Dawley rats. The optimal concentration of aldosterone to stimulate cardiac fibroblast proliferation was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cardiac fibroblasts were pretreated with 7.57 × 10(-4) mol/L or 3.78 × 10(-4) mol/L oxymatrine or without oxymatrine for 2 h, and then coincubated with 1 × 10(-8) mol/L aldosterone for 48 h. The MTT assay and Masson staining were used to detect the cardiac fibroblast proliferation and myofibroblast differentiation. The secretion of type I and III collagen was measured by commercial ELISA kits, and the hydroxyproline content was determined by the colorimetric assay. Western blotting assayed the Smad-2, Smad-3, and Smad-4 protein expression in cardiac fibroblasts. RESULTS: The present results confirmed that aldosterone induced cardiac fibroblast to myofibroblast proliferation and differentiation. The MTT assay and Masson staining indicated oxymatrine significantly inhibited aldosterone-induced cardiac fibroblast proliferation and myofibroblast differentiation. Oxymatrine significantly inhibited aldosterone-induced secretion of type I and III collagen, as indicated by commercial ELISA kits, and aldosterone-induced increase in hydroxyproline content, as indicated by a colorimetric assay. Western blotting revealed oxymatrine attenuated aldosterone-induced Smad-2, Smad-3, and Smad-4 expression in cardiac fibroblasts. CONCLUSION: Oxymatrine can inhibit cardiac fibroblast proliferation and differentiation into myofibroblasts via a mechanism linked to attenuation of the Smad signaling pathway.

Intercellular variation in signaling through the TGF-ß pathway and its relation to cell density and cell cycle phase.

Fundamental open questions in signal transduction remain concerning the sequence and distribution of molecular signaling events among individual cells. In this work, we have characterized the intercellular variability of transforming growth factor ß-induced Smad interactions, providing essential information about TGF-ß signaling and its dependence on the density of cell populations and the cell cycle phase. By employing the recently developed in situ proximity ligation assay, we investigated the dynamics of interactions and modifications of Smad proteins and their partners under native and physiological conditions. We analyzed the kinetics of assembly of Smad complexes and the influence of cellular environment and relation to mitosis. We report rapid kinetics of formation of Smad complexes, including native Smad2-Smad3-Smad4 trimeric complexes, in a manner influenced by the rate of proteasomal degradation of these proteins, and we found a striking cell to cell variation of signaling complexes. The single-cell analysis of TGF-ß signaling in genetically unmodified cells revealed previously unknown aspects of regulation of this pathway, and it provided a basis for analysis of these signaling events to diagnose pathological perturbations in patient samples and to evaluate their susceptibility to drug treatment.

TGF-ß1, Smad-2/-3, Smad-1/-5/-8, and Smad-4 signaling factors are expressed in ameloblastomas, adenomatoid odontogenic tumors, and calcifying cystic odontogenic tumors: an immunohistochemical study.

OBJECTIVES: The TGF-ß/Smad signaling pathway regulates diverse cellular functions, including tooth development, and is involved in numerous pathological processes such as tumorigenesis. The aim of this study was to investigate the immunoexpression of the TGF-ß/Smad signaling pathway members in ameloblastoma (AM), calcifying cystic odontogenic tumor (CCOT), and adenomatoid odontogenic tumor (AOT). MATERIALS AND METHODS: This retrospective cross-sectional study included 65 tissue specimens: 34 AMs, 13 CCOTs, and 18 AOTs. Serial sections were immunohistochemically stained with TGF-ß1, Smad-4, Smad-1/-5/-8, and Smad-2/-3 antibodies, and a semiquantitative measurement of the positive cells was carried out by two oral pathologists using a 0-3 scale (0: no immunoreactivity, 1: <20% positive cells, 2: 20-50% positive cells, 3: >50% positive cells). RESULTS: All biomarkers studied were found significantly decreased in AM compared to CCOT and AOT. AOT and CCOT expressed Smad-1/-5/-8 more strongly compared to AM (OR = 11.66, P < 0.001 and OR = 5.34, P = 0.013, respectively), and Smad-2/-3 immunostaining was found significantly increased in CCOT (OR = 10.42, P = 0.001) and AOT (OR = 5.16, P < 0.004) compared to AM. Similarly, Smad-4 was expressed more strongly in AOT and CCOT compared to AM (P = 0.001), while AOT demonstrated a fivefold higher chance to express TGF-ß1 compared to AM (P = 0.011). CONCLUSION: TGF-ß/Smad signaling pathway is activated in AM, AOT, and CCOT. The statistically significant reduced TGF-ß1/Smad immunoexpression in AM compared to AOT/CCOT could be associated with the more aggressive biological behavior of AM including increased cell proliferation and reduced apoptosis and differentiation. Thus, the biomarkers TGF-ß, Smad-4, Smad-1/-5/-8, and Smad-2/-3 could serve as supplementary diagnostic indices between odontogenic tumors of high and low neoplastic dynamics.

Levels of mRNA for bone morphogenetic proteins, their receptors and SMADs in goat ovarian follicles grown in vivo and in vitro.

This study investigated the stability of housekeeping genes (glyceraldehyde-3-phosphate dehydrogenase, ß-tubulin, ß-actin, phosphoglycerate kinase (PGK), 18S rRNA, ubiquitin and ribosomal protein 19) and the levels of mRNA for bone morphogenetic protein-2 (BMP-2), -4 (BMP-4), -6 (BMP-6), -7 (BMP-7) and -15 (BMP-15), their receptors (BMPR-IA, -IB and -II) and Similar to Mothers Against Decapentaplegic (SMADs) (-1, -5 and -8) in goat follicles of 0.2, 0.5 and 1.0mm, as well as in secondary follicles before and after culture for 18 days. ß-tubulin and PGK were the most stable housekeeping genes and the levels of mRNA for BMP-2 in follicles of 0.2mm were higher than in follicles of 0.5 and 1.0mm. For BMP-4, -6 and -7, the highest levels of mRNA were found in follicles of 1.0mm. The expression of BMPR-IB was higher in follicles of 0.2mm, whereas the levels of BMPR-II were higher in follicles of 0.5mm. The levels of mRNA for SMAD-5 were higher in follicles of 0.2mm, whereas SMAD-8 had higher levels in 0.5-mm follicles. After culture, follicles showed increased levels of mRNA for BMP-2 and reduced mRNA for BMP-4, BMP-7, BMPR-IA and SMAD-5. In conclusion, ß-tubulin and PGK are the most stable reference genes, and BMPs, their receptors and SMADs have variable levels of mRNA in the follicular size classes analysed.

Estrogen and antiestrogens alter breast cancer invasiveness by modulating the transforming growth factor-ß signaling pathway.

In the later stages of breast cancer, estrogen receptor (ER)α-negative cancers typically have higher histological grades than ERα-positive cancers, and transforming growth factor (TGF)-ß promotes invasion and metastasis. Our previous study indicated that ERα inhibited TGF-ß signaling by inducing the degradation of Smad in an estrogen-dependent manner. In the present study, we report that the suppressive effects of ERα and estrogen on tumor progression are mediated by inhibiting TGF-ß signaling. Furthermore, we investigated the effects of antiestrogens such as ICI182,780 (ICI) or tamoxifen (TAM) on TGF-ß signaling and breast cancer invasiveness. The levels of total Smad and pSmad were reduced by estrogen, whereas ICI slightly increased them, and TAM had no effect. To investigate the effect of antiestrogens on breast cancer invasiveness, we generated highly migratory and invasive MCF-7-M5 cells. The migration and invasion of these cells were suppressed by the inhibitor of TGF-ß receptor kinase, SB-505124, and estrogen. However, antiestrogens did not suppress the migration and invasion of these cells. In addition, we screened TGF-ß target genes whose expression was reduced by estrogen treatment and identified four genes associated with breast cancer invasiveness and poor prognosis. The expression of these genes was not decreased by antiestrogens. These observations provide a new insight into estrogen function and the mechanisms underlying estrogen-mediated suppression of tumor progression.

Myostatin levels in skeletal muscle of hibernating ground squirrels.

Myostatin, a negative regulator of muscle mass, is elevated during disuse and starvation. Mammalian hibernation presents a unique scenario, where animals are hypocaloric and in torpor, but the extent of muscle protein loss is minimized. We hypothesized that myostatin expression, which is usually increased early in disuse and under hypocaloric conditions, could be suppressed in this unique model. Skeletal muscle was collected from thirteen-lined ground squirrels, Spermophilus tridecemlineatus, at six time points during hibernation: control euthermic (CON); entrance into hibernation (ENT), body temperature (T(b)) falling; early hibernation (EHib), stable T(b) in torpor for 24 h; late hibernation (LHib), stable T(b) in torpor for 3 days; early arousal (EAr), T(b) rising; and arousal (AR), T(b) restored to 34-37°C for about 18 h. There was no significant increase of myostatin during ENT, EHib or LHib. Unexpectedly, there were approximately sixfold increases in myostatin protein levels as squirrels arose from torpor. The elevation during EAr remained high in AR, which represented an interbout time period. Mechanisms that could release the suppression or promote increased levels of myostatin were assessed. SMAD2 and phosphorylated SMAD2 were increased during EHib, but only the phosphorylated SMAD2 during AR mirrored increases in myostatin. Follistatin, a negative regulator of myostatin, did not follow the same time course as myostatin or its signaling pathway, indicating more control of myostatin at the signaling level. However, SMAD7, an inhibitory SMAD, did not appear to play a significant role during deep hibernation. Hibernation is an excellent natural model to study factors involved in the endogenous intracellular mechanisms controlling myostatin.

Bone morphogenic protein 2 directly enhances differentiation of murine osteoclast precursors.

Previous studies found that bone morphogenic proteins (BMPs) support osteoclast formation, but it is not clear whether this is a direct effect on osteoclasts or mediated indirectly through osteoblasts. We have shown that a mouse deficient for the BMP antagonist Twisted gastrulation suggested a direct positive role for BMPs on osteoclastogenesis. In this report, we further determine the significance of BMP signaling on osteoclast formation in vitro. We find that BMP2 synergizes with suboptimal levels of receptor activator of NF-kappaB ligand (RANKL) to enhance in vitro differentiation of osteoclast-like cells. The enhancement by BMP2 is not a result of changes in the rate of proliferation or survival of the bone marrow-derived cultures, but is accompanied by an increase in expression of genes involved in osteoclast differentiation and fusion. Treatment with BMP2 did not significantly alter expression of RANKL or OPG in our osteoclast cultures, suggesting that the enhancement of osteoclastogenesis is not mediated indirectly through osteoblasts or stromal cells. Consistent with this, we detected phosphorylated SMAD1,5,8 (p-SMAD) in the nuclei of mononuclear and multinucleated cells in osteoclast cultures. Levels of p-SMAD, BMP2, and BMP receptors increased during differentiation. RNAi suppression of Type II BMP receptor inhibited RANKL-stimulated formation of multinuclear TRAP-positive cells. The BMP antagonist noggin inhibited RANKL-mediated osteoclast differentiation when added prior to day 3, while addition of noggin on day 3 or later failed to inhibit their differentiation. Taken together, these data indicate that osteoclasts express BMP2 and BMP receptors, and that autocrine BMP signaling directly promotes the differentiation of osteoclasts-like cells.

Increased expression of Smad proteins, and in particular Smad3, in oral lichen planus compared to normal oral mucosa.

BACKGROUND: Oral lichen planus (OLP) is a chronic inflammatory disease of the oral mucosa which the World Health Organisation (WHO) considers a premalignant condition. One step in malignant development is so called epithelial mesenchymal transition (EMT), a process whereby epithelial cells acquire mesenchymal characteristics. EMT occurs during embryogenesis and wound healing but also in some human diseases such as cancer and fibrosis. A factor known to induce EMT is transforming growth factor-ß (TGF-ß), which uses the Smad proteins as mediators for its signalling. TGF-ß is also often over-expressed in squamous cell carcinoma of the head and neck (SCCHN). METHODS: In the present study we mapped expression of Smad proteins in OLP lesions by immunohistochemistry, and compared to expression in normal and sensitive oral mucosa. The latter group of patients had developed SCCHN after shorter or longer periods of diffuse oral symptoms. The aim was to see if there were any signs of EMT related changes in the OLP lesions, as judged by changes in the TGF-ß pathway. CONCLUSION: Changes in the TGF-ß pathway related to EMT are seen in the very earliest stages of oral malignancy and become more severe as lesions progress.

Surface nanofeature effects on titanium-adherent human mesenchymal stem cells.

PURPOSE: Hydrofluoric acid treatment of moderately rough commercially pure titanium produced by titanium oxide (TiO2) grit blasting (OsseoSpeed) results in a surface with nanofeatures. The aim of this project was to better understand the effect of surface nanotopography on adherent osteoblastic differentiation. MATERIALS AND METHODS: Human mesenchymal stem cells were grown on TiO2 grit-blasted and hydrofluoric acid-treated/TiO2 grit-blasted titanium coins for 1 to 28 days. The nature of the surfaces was evaluated using scanning electron microscopy, optical interferometry, and x-ray photoelectron spectrometry. Osteoblastic differentiation was measured using real-time polymerase chain reaction measurement of more than 80 mineralized tissue-associated, protein-encoding mRNAs. RESULTS: Hydrofluoric acid-treated surfaces displayed nanofeatures of 100 nm in diameter and maintenance of micron-level topography. Adherent cell osteoblastic differentiation occurred on both surfaces but took place more rapidly and to a greater extent on hydrofluoric acid-treated surfaces. This was revealed by earlier, higher, and sustained levels of osteoinductive transcription factors (RUNX-2, SMADs), growth factors (insulin-like growth factor 2, bone morphogenetic proteins), and bone matrix proteins. CONCLUSIONS: The superimposition of nanofeatures on a moderately rough commercially pure titanium surface is associated with marked osteoinduction and osteogenesis of adherent mesenchymal stem cells. The role of nanotopography in directing adherent cell behavior should be fully investigated.

Distribution of Smad mRNA and proteins in the rat brain.

Smad proteins are known to transduce the action of TGF-ß superfamily proteins including TGF-ßs, activins, and bone morphogenetic proteins (BMPs). In this study, we examined the expression of Smad1, -2, -3, -4, -5, and -8 mRNA in the rat brain by means of RT-PCR and in situ hybridization (ISH). In addition, we examined the nuclear accumulation of Smad1, -2, -3, -5, and -8 proteins after intracerebroventricular injection of TGF-ß1, activin A, or BMP6 with immunohistochemistry to investigate whether TGF-ß, activin, and/or BMP activate Smads in the rat brain. RT-PCR analysis revealed that Smad1, -2, -3, -4, -5, and -8 mRNA was expressed in the brain and that the Smad3 and Smad8 mRNA differed in the expression level between brain regions. For example, there were high levels of expression of Smad3 mRNA in the cerebral cortex, caudate putamen/globus pallidus, and cerebellum, but low levels in the thalamus and midbrain. Expression of Smad8 mRNA was higher in the midbrain, cerebellum, and pons/medulla oblongata in comparison to the olfactory bulb, cerebral cortex, caudate putamen/globus pallidus, hippocampus/dentate gyrus, and thalamus. ISH signals for Smad1 mRNA were widely detected in the brain except for a small number of regions including the olfactory tubercle, posterior region of hypothalamus, and cerebellar nuclei. ISH signals for Smad2 mRNA were abundantly observed in several brain regions including the olfactory bulb, piriform cortex, basal ganglia, cingulate cortex, epithalamus, including the pineal gland and medial habenular nuclei, hypothalamus, inferior colliculi of the midbrain, and some nuclei in the pons, cerebellar cortex, and choroid plexus. ISH signals for Smad3 mRNA were also abundantly observed in several brain regions. Especially strong signals for Smad3 mRNA were observed in the olfactory tubercle, piriform cortex, basal ganglia, dentate gyrus, and cingulate cortex. ISH signals for Smad5 and Smad8 mRNA were restricted to a small number of brain regions, the signal intensity of which was weak. ISH signals for Smad4 mRNA were detected in all regions examined. Intracerebroventricular injection of activin A induced nuclear accumulation of Smad2 and Smad3 immunoreactivity in neurons. In contrast, intracerebroventricular injection of TGF-ß1 or BMP6 did not induce nuclear accumulation of the immunoreactivity for any Smad in neurons. These results suggest that activin-Smad signaling plays an important role in brain homeostasis.

Immunolocalization of Advanced Glycation End Products, Mitogen Activated Protein Kinases, and Transforming Growth Factor-ß/Smads in Pelvic Organ Prolapse.

Collagen and matrix metalloproteinases (MMP) play a pivotal role in the pathophysiology of Pelvic Organ Prolapse (POP) as a switch between type I and III collagen together with a simultaneous activation of MMPs have been observed in the vaginal wall. The aim of this study was to evaluate the Advanced Glycation End (AGE) products, ERK1/2 and transforming growth factor (TGF)-ß/Smad pathway expression in muscularis propria in women with POP compared with control patients. We examined 20 patients with POP and 10 control patients treated for uterine fibromatosis. Immunohistochemical analysis using AGE, RAGE, ERK1/2, Smads-2/3, Smad-7, MMP-3, and collagen I-III, TIMP, and α-SMA were performed. Smad-2/3, Smad-7, AGE, ERK1/2, p-ERK, and p-Smad3 were also evaluated using Western-blot analysis. POP samples from the anterior vaginal wall showed disorganization of the normal muscularis architecture. In POP samples, AGE, ERK1/2, Smad-2/3, MMP-3, and collagen III were upregulated in muscularis whereas in controls, Smad-7 and collagen I were increased. The receptor for AGEs (RAGE) was mild or absent both in controls and prolapse. We demonstrated the involvement of these markers in women with POP but further studies are required to elucidate if the overexpression of these molecules could play a crucial role in the pathophysiology of POP disease.

[Effect of acupuncture on TGF-ß1/Smads pathway in mice with airway remodeling mic].

OBJECTIVE: To investigate the effect of acupuncture on TGF-ß1/Smads signaling pathway in the lung tissue of mice with airway remodeling. METHODS: Thirty specific pathogen-free mice were randomly divided into blank group, model group and acupuncture group (n=10). Mouse models of asthma were established in the model group and the acupuncture group, and the mice in the latter group received 7 acupuncture therapies (at bilateral Fei Shu, Da Zhui and Zu Sanli, 20 min each time) every other day, starting on the 10th day after the modeling. At 24 h after the last acupuncture, the mice were subjected to inhalation of 1% OVA for 3 days, and 24 h after the last challenge, the mice were given methacholine chloride (Mch) inhalation at different concentrations for measurement of lung resistance using a noninvasive stroke volume meter. HE staining was used to observe the pathological changes in the lung tissues, and TGF-ß1 levels in the the bronchoalveolar lavage fluid (BALF) and serum were detected using ELISA; Western blotting was used to detect the differential protein expressions in the airway smooth muscles between the two groups. The airway smooth muscle cells were isolated from the mice in the acupuncture group and treated with a TGF- ß1 inhibitor (LY2157299), and the relative expressions of type-Ⅰ and Smads proteins were detected using Western blotting. RESULTS: The mice in the model showed obvious tracheal fistula with airway pathologies including lumen narrowing, bronchial mucosa thickening, dissociation of the epithelial cells, and thickening of the alveolar septum and airway smooth muscles. These pathological changes were obviously milder in the acupuncture group. The asthmatic mice exhibited significantly increased lung resistance in positive correlation with Mch concentration. Serum TGF-ß1 level was significantly elevated in asthmatic mice (P < 0.05); TGF-ß1 levels in the serum and BALF were significantly lower in the acupuncture group than in the model group (P < 0.05). In the model group, the expressions of α-SMA, TGF-ß1 and Smads in the airway smooth muscles were significantly higher than those in the other two groups (both P < 0.05). In cultured airway smooth muscle cells, the expressions of type-Ⅰ and Smads were significantly higher in cells treated with LY2157299 than in the control cells (P>0.05). CONCLUSIONS: Acupuncture can inhibit airway remodeling by inhibiting the expression of airway TGF-ß1 and down-regulating the expression of Smads and α-SMA to reduce airway inflammatory response. Airway expressions of type-Ⅰ and Smads proteins remain high after inhibiting TGF-ß1. Acupuncture may control asthma progression through the TGF-ß1/Smads pathway.

Role of transforming growth factor ß­1 in the pathogenesis of pelvic organ prolapse: A potential therapeutic target.

The present study aimed to reveal the metabolic alterations of the extracellular matrix (ECM) in uterosacral ligament (USL) with pelvic organ prolapse (POP) and to explore the role of transforming growth factor­ß1 (TGF­ß1) in pathogenesis of POP. For this purpse, 60 participants who underwent hysterectomy for benign indications were enrolled, 30 of which had symptomatic POP (grade II, III or IV) and composed the POP group, and the other 30 had asymptomatic POP (grade I or less) and served as the controls. Collagen fibers, elastin,matrix metalloproteinase (MMP)­2/9, tissue inhibitor of matrix metalloproteinases (TIMP)­2 and TGF­ß1 were examined by Masson's trichrome staining, immunohistochemistry and RT-qPCR using USL biopsies. In vitro, human USL fibroblasts (hUSLFs) were primary cultured, pre-treated with recombinant TGF­ß1 (0, 5, or 10 ng/ml) and then subjected to cyclic mechanical stretching (CMS; 0 or 5,333 µÎµ strain). Changes in the expression levels of collagen type I/III, elastin, TIMP­2, MMP­2/9 and Smad were detected. Our results revealed that at the tissue level, the expression of collagen fibers, elastin, TIMP­2 and TGF­ß1 was significantly reduced in the POP group, while the activities of MMP­2/9 were significantly upregulated, compared with the control group. Statistical analysis indicated that the mRNA expression of TGF­ß1 inversely correlated with the severity of POP partially. Our in vitro experimental data demonstrated that a CMS of 5333 µÎµ strain promoted the degradation of ECM proteins, inhibited the synthesis of TIMP­2, and upregulated the proteolytic activities of MMP­2/9. Pre-treatment with TGF­ß1 attenuated the loss of ECM by stimulating the synthesis of TIMP­2 and inhibiting the activities of MMP­2/9 through the TGF­ß1/Smad3 signaling pathway. On the whole, our data indicate that the reduced anabolism and increased catabolism of ECM proteins in USL are the pathological characteristics of POP. TGF­ß1 not only has a specific value in predicting the severity of POP, but should also be considered as a novel therapeutic target for POP.

Transforming growth factor-ß1 SMAD effectors and medial cell number in ascending aorta diseases.

In ascending aorta aneurysms and dissections, the extracellular matrix is degraded. Transforming growth factor (TGF)-ß1 modulates its synthesis. The production and presence of SMADs, intracellular effectors of TGF-ß1 signaling, were analyzed in patients with these diseases. To verify whether medial cells are lost, their total numbers were computed. Ascending aorta samples from 19 patients and 18 controls underwent immunoperoxidase reactions to SMADs 2, 3, 4, and 7. Positive and negative cells were counted, and total numbers of cells and positive/total ratios were calculated. Samples from other 14 patients and 7 normal controls were used for the quantification of SMAD3, SMAD4, and SMAD7 mRNA. For SMAD4, both mRNA (2.36 vs. 0.37, P=.03) and ratio of positive cells (0.94 vs. 0.73, P=.02) are increased in patients with ascending aortic diseases. SMAD3 mRNA was also increased (1.19 vs. 0.20, P=.05). The cell ratios of this and the other SMADs, SMAD7 mRNA, and the total cell count did not differ between groups. In conclusion, in ascending aortic aneurysms and dissections, there is an increase in SMAD4, implicated in extracellular matrix production, possibly as an attempt to compensate for extracellular matrix deficiency. Lost medial cells are replaced, since their number is not diminished.

Multiple myeloma causes clonal T-cell immunosenescence: identification of potential novel targets for promoting tumour immunity and implications for checkpoint blockade.

Tumour-induced dysfunction of cytotoxic T cells in patients with multiple myeloma (MM) may contribute to immune escape and be responsible for the lack of therapeutic efficacy of immune checkpoint blockade. We therefore investigated dysfunctional clonal T cells in MM and demonstrated immunosenescence but not exhaustion as a predominant feature. T-cell clones were detected in 75% of MM patients and their prognostic significance was revalidated in a new post-immunomodulatory drug cohort. The cells exhibited a senescent secretory effector phenotype: KLRG-1+/CD57+/CD160+/CD28-. Normal-for-age telomere lengths indicate that senescence is telomere independent and potentially reversible. p38-mitogen-activated protein kinase, p16 and p21 signalling pathways known to induce senescence were not elevated. Telomerase activity was found to be elevated and this may explain how normal telomere lengths are maintained in senescent cells. T-cell receptor signalling checkpoints were normal but elevated SMAD levels associated with T-cell inactivation were detected and may provide a potential target for the reversal of clonal T-cell dysfunction in MM. Low programmed death 1 and cytotoxic T-lymphocyte-associated antigen 4 expression detected on T-cell clones infers that these cells are not exhausted but suggests that there would be a suboptimal response to immune checkpoint blockade in MM. Our data suggest that other immunostimulatory strategies are required in MM.

Inactivated Pseudomonas aeruginosa inhibits hypoxia-induced pulmonary hypertension by preventing TGF-ß1/Smad signaling.

Pseudomonas aeruginosa is one of the common colonizing bacteria of the human body and is an opportunistic pathogen frequently associated with respiratory infections. Inactivated P. aeruginosa (IPA) have a variety of biological effects against inflammation and allergy. Transforming growth factor-ß (TGF-ß) signaling plays a critical role in the regulation of cell growth, differentiation, and development in a wide range of biological systems. The present study was designed to investigate the effects of IPA on TGF-ß/Smad signaling in vivo, using a hypoxia-induced pulmonary hypertension (PH) rat model. Sprague Dawley rats (n=40) were exposed to 10% oxygen for 21 days to induce PH. At the same time, IPA was administered intravenously from day 1 to day 14. Mean pulmonary artery pressure (mPAP) and the right ventricle (RV) to left ventricle plus the interventricular septum (LV+S) mass ratio were used to evaluate the development of PH. Vessel thickness and density were measured using immunohistochemistry. Primary arterial smooth muscle cells (PASMCs) were isolated and the proliferation of PASMCs was assayed by flow cytometry. The production of TGF-ß1 in cultured supernatant of PASMCs was assayed by ELISA. The expression levels of α-smooth muscle actin (α-SMA), TGF-ß1 and phospho-Smad 2/3 in PASMCs were assayed by western blot. Our data indicated that IPA attenuated PH, RV hypertrophy and pulmonary vascular remodeling in rats, which was probably mediated by restraining the hypoxia-induced overactive TGF-ß1/Smad signaling. In conclusion, IPA is a promising protective treatment in PH due to the inhibiting effects on TGF-ß1/Smad 2/3 signaling.