Investigation of the Antihypertrophic and Antifibrotic Effects of Losartan in a Rat Model of Radiation-Induced Heart Disease.

Radiation-induced heart disease (RIHD) is a potential late side-effect of thoracic radiotherapy resulting in left ventricular hypertrophy (LVH) and fibrosis due to a complex pathomechanism leading to heart failure. Angiotensin-II receptor blockers (ARBs), including losartan, are frequently used to control heart failure of various etiologies. Preclinical evidence is lacking on the anti-remodeling effects of ARBs in RIHD, while the results of clinical studies are controversial. We aimed at investigating the effects of losartan in a rat model of RIHD. Male Sprague-Dawley rats were studied in three groups: (1) control, (2) radiotherapy (RT) only, (3) RT treated with losartan (per os 10 mg/kg/day), and were followed for 1, 3, or 15 weeks. At 15 weeks post-irradiation, losartan alleviated the echocardiographic and histological signs of LVH and fibrosis and reduced the overexpression of chymase, connective tissue growth factor, and transforming growth factor-beta in the myocardium measured by qPCR; likewise, the level of the SMAD2/3 protein determined by Western blot decreased. In both RT groups, the pro-survival phospho-AKT/AKT and the phospho-ERK1,2/ERK1,2 ratios were increased at week 15. The antiremodeling effects of losartan seem to be associated with the repression of chymase and several elements of the TGF-ß/SMAD signaling pathway in our RIHD model.

Prognostic Significance of TLR2, SMAD3 and Localization-dependent SATB1 in Stage I and II Non-Small-Cell Lung Cancer Patients.

This study aimed to explore the prognostic value of SATB1, SMAD3, and TLR2 expression in non-small-cell lung carcinoma patients with clinical stages I-II. To investigate, we evaluated immunohistochemical staining to each of these markers using tissue sections from 69 patients from our cohort and gene expression data for The Cancer Genome Atlas (TCGA) cohort. We found that, in our cohort, high expression levels of nuclear SATB1n and SMAD3 were independent prognostic markers for better overall survival (OS) in NSCLC patients. Interestingly, expression of cytoplasmic SATB1c exhibited a significant but inverse association with survival rate, and it was an independent predictor of unfavorable prognosis. Likewise, TLR2 was a negative outcome biomarker for NSCLC even when adjusting for covariates. Importantly, stratification of NSCLCs with respect to combined expression of the three biomarkers allowed us to identify subgroups of patients with the greatest difference in duration of survival. Specifically, expression profile of SATB1n-high/SMAD3high/TLR2low was associated with the best OS, and it was superior to each single protein alone in predicting patient prognosis. Furthermore, based on the TCGA dataset, we found that overexpression of SATB1 mRNA was significantly associated with better OS, whereas high mRNA levels of SMAD3 and TLR2 with poor OS. In conclusion, the present study identified a set of proteins that may play a significant role in predicting prognosis of NSCLC patients with clinical stages I-II.

Adipocytokine expression, platelet-to-lymphocyte ratio and TGF-ß1/Smad signaling activity in diabetic patients complicated with pulmonary infection.

OBJECTIVE: To investigate adipocytokine expression levels, platelet-to-lymphocyte ratio (PLR) and transforming growth factor (TGF)-ß1/Smad signaling activity in diabetic patients with pulmonary infection. METHODS: Eighty-two type 2 diabetic patients with pulmonary infection were included in the observation group and 75 patients with simple type 2 diabetes were recruited into the control group. The fasting blood glucose (FBG), glycated hemoglobin (HbA1c), and PLR in the two groups were compared. Complement-C1q/tumor necrosis factor related protein 3 (CTRP-3), complement-C1q/tumor necrosis factor related protein 9 (CTRP-9), leptin, adiponectin, and TGF-ß1/Smad signaling pathway activity in peripheral blood mononuclear cells (PBMCs) was detected. RESULTS: Compared with patients in the control group, patients in the observation group presented with increased levels of FGB, HbA1c, and leptin, an increase in the PLR, and decreased serum CTRP-3, CTRP-9, and adiponectin levels. TGF-ß1, p-Smad2, and p-Smad3 protein expression levels were up-regulated in PBMCs from patients in the observation group compared with the control group. CONCLUSIONS: These results show that in type 2 diabetic patients with pulmonary infection, adipocytokine expression is altered, PLR is disturbed, and the TGF-ß1/Smad signaling pathways in PBMCs are significantly activated.

Role of phosphorylated Smad3 signal components in intraductal papillary mucinous neoplasm of pancreas.

BACKGROUND: Malignant intraductal papillary mucinous neoplasm (IPMN) has poor prognosis. The carcinogenesis of IPMN is not clear. The aim of this study was to clarify transitions in phosphorylated Smad3 signaling during IPMN carcinogenesis. METHODS: By using immunohistochemistry, we examined the expression of pSmad3C and pSmad3L from 51 IPMN surgical specimens resected at our institution between 2010 and 2013. We also examined the expression of Ki-67, c-Myc and p-JNK. RESULTS: The median immunostaining index of pSmad3C was 79.2% in low-grade dysplasia, 74.9% in high-grade dysplasia, and 42.0% in invasive carcinoma (P < 0.01), whereas that of pSmad3L was 3.4%, 4.3%, and 42.4%, respectively (P < 0.01). There was a negative relationship between the expression of pSmad3C and c-Myc (P < 0.001, r = -0.615) and a positive relationship between the expression of pSmad3L and c-Myc (P < 0.001, r = 0.696). Negative relationship between the expression of pSmad3C and Ki-67 (P < 0.01, r = -0.610) and positive relationship between the expression of pSmad3L and Ki-67 (P < 0.01, r = 0.731) were confirmed. p-JNK-positive cells were frequently observed among pSmad3L-positive cancer cells. The median of pSmad3L/pSmad3C ratio in the non-recurrence group and the recurrence group were 0.58 (range, 0.05-0.93), 3.83 (range, 0.85-5.96), respectively (P = 0.02). The median immunostaining index of c-Myc in the non-recurrence group and the recurrence group were 2.91 (range, 0-36.9) and 82.1 (range, 46.2-97.1), respectively (P = 0.02). The median immunostaining index of Ki-67 in the non-recurrence group and the recurrence group were 12.9 (range 5.7-30.8) and 90.9 (range 52.9-98.5), respectively (P = 0.02). CONCLUSIONS: pSmad3L was upregulated in malignant IPMN. pSmad3L/pSmad3C ratio may be a useful prognostic factor in IPMN.

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.

c­Jun N­terminal kinase/transforming growth factor­ß/Smad3 pathway: Is it associated with endoplasmic reticulum stress­mediated renal interstitial fibrosis?

The present study investigated the role of the c­Jun N­terminal kinase (JNK)/transforming growth factor­ß (TGF­ß)/Smad3 pathway in endoplasmic reticulum stress (ERS)­mediated renal interstitial fibrosis, which would be beneficial for chronic kidney disease (CKD) therapy. In human renal biopsy tissue, the expression levels of glucose­regulated protein 78 (GRP78) and phosphorylated (p)­JNK were examined by immunohistochemical analysis. In renal tubular HK­2 cells, tunicamycin (TM) was used to induce ERS, and the cells were then treated with the chemical ERS inhibitor 4­phenylbutyrate (4­PBA) or the chemical JNK pathway inhibitor SP600125, respectively. Western blotting was then performed in the cells to determine the expression levels of GRP78 and p­JNK proteins, as well as TGF­ß/Smad3 pathway­associated proteins, including TGF­ß1, p­Smad3, connective tissue growth factor and α­smooth muscle actin. The results revealed that GRP78 and p­JNK were evidently expressed in the renal tissues of patients with CKD, and these expression levels were significantly higher in renal tissues with severe interstitial fibrosis compared with glomerular minor lesion tissues (P<0.01 and P<0.05, respectively). Furthermore, ERS and JNK pathway inhibition decreased the expression levels of TGF­ß/Smad3 pathway signals in cells incubated with TM. ERS pathway inhibition also attenuated the expression levels of p­JNK in HK­2 cells. In conclusion, ERS was observed to serve an important role in the pathogenesis of CKD and may induce renal interstitial fibrosis via the JNK/TGF­ß/Smad3 pathway.

Genomic Observations of a Rare/Pathogenic SMAD3 Variant in Loeys⁻Dietz Syndrome 3 Confirmed by Protein Informatics and Structural Investigations.

Background and objectives: Loeys-Dietz syndrome 3, also known as aneurysms--osteoarthritis syndrome, is an autosomal dominant genetic connective tissue disease caused by pathogenic variants in SMAD3, a transcription factor involved in TGF-ß signaling. This disorder is characterized by early-onset osteoarthritis and arterial aneurysms. Common features include scoliosis, uvula abnormalities, striae, and velvety skin. Materials and Methods: The pathogenicity of a variant of uncertain significance in the SMAD3 gene was evaluated (variant c.220C > T) through personalized protein informatics and molecular studies. Results: The case of a 44-year-old male, who was originally presumed to have Marfan syndrome, is presented. An expanded gene panel determined the probable cause to be a variant in SMAD3, c.220C > T (p.R74W). His case was complicated by a history of stroke, but his phenotype was otherwise characteristic for Loeys-Dietz syndrome 3. Conclusion: This case emphasizes the importance of comprehensive genetic testing to evaluate patients for connective tissue disorders, as well as the potential benefit of utilizing a protein informatics platform for the assessment of variant pathogenicity.

Protective effect of amygdalin on epithelial-mesenchymal transformation in experimental chronic obstructive pulmonary disease mice.

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory pulmonary disease characterized by continuous, progressive limitation of airflow. Airway remodelling, which is correlated with epithelial-mesenchymal transitions (EMTs), is a typical pathophysiological change of COPD. Amygdalin, an active ingredient in the traditional Chinese medicine bitter almond with extensive pharmacological effects, was shown to inhibit tissue fibrosis in recent studies. In this study, a human bronchial epithelial cell line (BEAS-2B) and mice were exposed to cigarette smoke, and EMT levels were investigated after treatment with different concentrations of amygdalin. Morphology was assessed by immunohistochemical staining. Evaluation of the expression of TGF-ß1, smad2/3, and p-smad2/3 in lung tissue was conducted out via ELISA, Western blot, and real-time PCR. The results showed that E-cadherin expression was significantly increased, whereas vimentin, TGF-ß1, and phosphorylated smad2/3 (p-smad2/3) expression was markedly decreased in the amygdalin-treated groups compared with the model group. Therefore, our study demonstrated a protective role of amygdalin in the murine EMT process after COPD.

Downregulation of TGF-ß1 suppressed proliferation and increased chemosensitivity of ovarian cancer cells by promoting BRCA1/Smad3 signaling.

BACKGROUND: Studies have demonstrated that transforming growth factor beta-1 (TGF-ß1) exhibits oncogenic activity in different types of cancer, including ovarian cancer (OC). However, its regulatory mechanism in OC and whether TGF-ß1 is involved in chemosensitivity regulation remains unclear. Thus, the aim of this study was to investigate the role of TGF-ß1 in OC. METHODS: The OC cell line SKOV3 was employed, and TGF-ß1 overexpression or knockdown vectors were constructed. The cell proliferation of SKOV3 was evaluated with the cell counting kit (CCK8) kit after treatment with different concentrations of cis-platinum. Western blot and protein immunoprecipitation were employed to detect changes in BRCA1 and Smad3 expression and their interactions. Tumor growth in nude mice was evaluated. RESULTS: The results showed that TGF-ß1 knockdown increased chemosensitivity by promoting BRCA1 expression and Smad3 phosphorylation. In vivo studies showed that TGF-ß1 knockdown significantly inhibited the growth of tumors, also by upregulating BRCA1 expression and Smad3 phosphorylation. CONCLUSION: Taken together, our results suggest that TGF-ß1 knockdown inhibits tumor growth and increases chemosensitivity by promotion of BRCA1/Smad3 signaling.

Targeting newly identified ERß/TGF-ß1/SMAD3 signals with the FDA-approved anti-estrogen Faslodex or an ERß selective antagonist in renal cell carcinoma.

Renal cell carcinoma (RCC) has the third highest mortality rate among urological tumors, and 20-30% of RCC patients present with metastatic RCC at the time of diagnosis. Although recent studies have indicated that estrogen receptor ß (ERß) could play promoting roles in RCC progression, the detailed mechanisms remain to be clarified. In the present study, we found that expression of ERß, but not ERα, increases with tumor stage and grade, and also observed that modification of ERß signals using estrogens/anti-estrogens, shRNA knockdown of ERß and overexpression of ERß using ectopic cDNA affects RCC cell proliferation, migration and invasion. Mechanism analysis revealed that ERß can promote RCC cell invasion via an increase in transforming growth factor ß1 (TGF-ß1)/SMAD3 signals, and interrupting TGF-ß1/SMAD3 signals with a TGFßR1 inhibitor can reverse/block ERß-increased RCC cell migration. Importantly, preclinical analyses using in vivo mouse models of RCC revealed that targeting of this newly identified ERß/TGF-ß1/SMAD3 pathway with either the FDA-approved anti-estrogen ICI182,780 (Faslodex) or a selective ERß antagonist 4-[2-phenyl-5,7 bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol can significantly reduce RCC tumor growth and invasion, which may be suitable as the basis for novel therapies to more effectively suppress metastatic RCC.

Potential ameliorative effects of epigallocatechin­3­gallate against testosterone-induced benign prostatic hyperplasia and fibrosis in rats.

Green tea is among the most popular beverages in the world and is an important source of phytoestrogens. Epigallocatechin­3­gallate (EGCG) is the major polyphenol in green tea. The aim of this study was to investigate the anti-benign prostatic hyperplasia (BPH) activity and underling mechanisms of EGCG in testosterone-induced BPH rats and in BPH-1 cells. Prostatic levels of oxidative stress and inflammation makers, as well as angiogenesis related growth factors were measured. Additionally, the prostatic levels of sex hormonal mediators (androgen receptor (AR), estrogen receptor (ER)-α and ER-ß), hypoxia-inducible factor (HIF)-1α, transforming growth factor-ß1 (TGF-ß1), type I TGF-ß receptor (TGF-ßRI), Smad3, phosphorylation-Smad3 (p-Smad3), epithelial-mesenchymal transition (EMT) markers (E-cadherin, collagen-I, fibronectin and α-SMA) and microRNA (miR)-133a/b were analyzed by immunohistochemistry assay, western blot and/or quantitative RT-PCR. It was observed that EGCG attenuated the prostatic oxidative stress and inflammatory microenvironment, ameliorated prostatic hyperplasia and collagen deposition, reduced the levels of angiogenesis related growth factors, inhibited the over-expression of AR, ER-α, HIF-1α, TGF-ß1, TGF-ßRI and p-Smad3, enhanced the expression of ER-ß, increased the levels of miR-133a/b, as well as relieved prostatic EMT in rats. Both HIF-1α inhibitor and EGCG decreased the expression of HIF-1α and TGF-ß1, as well as attenuated EMT in BPH-1 cells. It indicated that EGCG could attenuate testosterone-induced BPH and fibrosis.

SIS3, a specific inhibitor of smad3, attenuates bleomycin-induced pulmonary fibrosis in mice.

Pulmonary fibrosis (PF) is a fatal respiratory disease with no effective medical treatments available. TGF-ß/Smads signaling has been implicated to play an essential in the pathogenesis of PF, in which Smad3 act as the integrator of pro-fibrosis signals. In this study, we determined the effect of SIS3, a specific inhibitor of Smad3, in an experimental mouse model of lung fibrosis. We observed that SIS3 treatment significantly reduced bleomycin (BLM)-induced pathological changes and collagen deposition in the lung as indicated by Masson staining, real-time PCR and hydroxyproline content assay. As expected, the levels of Smad3 phosphorylation were decreased in the lung of mice treated with SIS3. Furthermore, SIS3 treatment also suppressed BLM-induced infiltration of inflammatory cells in the lung. Taken together, our results suggest that SIS3 ameliorated BLM-induced PF in mouse lungs. Thus, targeting Smad3 with SIS3 may be an effective approach for treatment of fibrotic disorders.

Human umbilical cord mesenchymal stem cells inhibit proliferation of hepatic stellate cells in vitro.

The effect of human umbilical cord mesenchymal stem cells (hUC-MSCs) on the proliferation of hepatic stellate cells (HSCs) is largely unknown. The purpose of this study was to explore the mechanism of action of hUC­MSCs on the proliferation of HSCs in vitro. The upper and lower double-cell co-culture system was established between hUC­MSCs and HSCs in the experimental group. HSCs were cultured alone as a negative control group. Cell proliferation and apoptosis were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively. Cell supernatants were harvested to determine the concentration of transforming growth factor-ß1 (TGF-ß1) by ELISA. mRNA and protein of TGF-ß1, Smad3 and Smad7 in HSCs were determined by reverse transcription-polymerase chain reaction and western blotting, respectively. In the co-culture group, the proliferation of HSCs was significantly inhibited compared with the negative control group at 24 and 48 h (p<0.05). Apoptosis of HSCs in the co-culture group increased compared with that in the negative control group, which was more obvious at 48 h (p<0.05). The concentration of TGF-ß1 in the co-culture group was significantly lower than in the HSCs cultured alone (p<0.05). After HSCs were co-cultured with hUC­MSCs for 48 h, expression of TGF-ß1 and Smad3 mRNA and protein was reduced and expression of Smad7 mRNA and protein was increased compared with the negative control group (p<0.05). hUC­MSCs inhibited proliferation of HSCs, possibly through inhibiting TGF-ß1 and Smad3 expression and increasing Smad7 protein expression.

TGF-ß1/TßRII/Smad3 signaling pathway promotes VEGF expression in oral squamous cell carcinoma tumor-associated macrophages.

Oral squamous cell carcinoma (OSCC) is the most common type of malignant cancer affecting the oral cavity. Tumor associated macrophages (TAMs) play a vital role in the initiation, progression and metastasis of OSCC. In this study, we investigated the correlation between macrophages and several clinical and pathological indicators, and we also explored how transforming growth factor-ß1 (TGF-ß1) effect on VEGF expression in TAMs. Seventy-two paraffin-embedded OSCC samples were collected. Association between macrophages density, micro vascular density (MVD) and clinical-pathological feature were explored by immunohistochemical staining. Western blot, ELISA and qRT-PCR were conducted to assess the VEGF expression in TAMs treated with or without neutralizing TGF-ß1, TßRII and smad3 antibodies. Results showed that CD68+ macrophages were absent in normal tissues. Macrophages density was directly correlated to low pathological differentiation, late clinical staging and poor survival rate. MVD showed positive correlation with clinical staging and macrophages density. Furthermore, OSCC-associated macrophages expressed more VEGF than macrophages in healthy lymph nodes. However, when TGF-ß1 or TßRII were neutralized or the Smad3 was inhibited, VEGF expression was down regulated as well. It is concluded that TGF-ß1 could promote OSCC-associated macrophages to secrete more VEGF via TßRII/Smad3 signaling pathway. This result might explain the correlation between macrophages density and worse clinical-pathological condition.

Effect of genistein on myocardial fibrosis in diabetic rats and its mechanism.

The aim of the present study was to investigate the effects of genistein (GEN) on myocardial fibrosis in type 1 diabetic rats and explore the underlying mechanisms. Rats were divided into 4 groups: Normal control (N), diabetic control (D), low­dose GEN treatment (L) and high­dose GEN treatment (H) groups. Following 8 weeks, the ventricular hemodynamic parameters, fasting blood glucose (FBG), heart­weight to body­weight ratio (HW/BW), myocardial hydroxyproline (Hyp) content, serum creatine kinase MB isozyme (CK­MB), lactate dehydrogenase (LDH), tumor necrosis factor­α (TNF­α), interleukin­1ß (IL­1ß) and interleukin­6 (IL­6) levels were measured. The histomorphology and ultrastructure of the heart were observed. The protein expression of myocardial transforming growth factor­ß1 (TGF­ß1), mothers against decapentaplegic homolog (Smad)­3, phosphorylated (p)­Smad3, Smad4, collagen­I and collagen­III were estimated. Compared with the N group, while the cardiac function was decreased, the levels of FBG, HW/BW, Hyp content, CK­MB, LDH, TNF­α, IL­1ß and IL­6 were increased in the D group. The myocardial histomorphological alterations and ultrastructure were damaged, and the protein expression of myocardial TGF­ß1, Smad3, p­Smad3, Smad4, collagen­I and collagen­III were increased in the D group. Compared with the D group, there were no differences in the ventricular hemodynamic parameters, FBG and p­Smad3 expression in the L group, while HW/BW, Hyp content, CK­MB, LDH, TNF­α, IL­1ß and IL­6 levels were decreased. The myocardial histomorphological damage was alleviated and the protein expression of TGF­ß1, Smad3, Smad4, collagen­I and collagen­III was decreased in the L group. Compared with L group, excluding FBG, the aforementioned indices were improved in the H group. In conclusion, GEN can attenuate myocardial fibrosis in type 1 diabetic rats, and the underlying mechanisms may be associated with the reduction of CK­MB and LDH leakage, inhibition of the inflammatory reaction, and suppression of the TGF­ß1/Smad3 signaling pathway to regulate collagen expression.

Downregulation of TGF-ß1 suppressed proliferation and increased chemosensitivity of ovarian cancer cells by promoting BRCA1/Smad3 signaling

BACKGROUND: Studies have demonstrated that transforming growth factor beta-1 (TGF-ß1) exhibits oncogenic activity in different types of cancer, including ovarian cancer (OC). However, its regulatory mechanism in OC and whether TGF-ß1 is involved in chemosensitivity regulation remains unclear. Thus, the aim of this study was to investigate the role of TGF-ß1 in OC. METHODS: The OC cell line SKOV3 was employed, and TGF-ß1 overexpression or knockdown vectors were constructed. The cell proliferation of SKOV3 was evaluated with the cell counting kit (CCK8) kit after treatment with different concentrations of cis-platinum. Western blot and protein immunoprecipitation were employed to detect changes in BRCA1 and Smad3 expression and their interactions. Tumor growth in nude mice was evaluated. RESULTS: The results showed that TGF-ß1 knockdown increased chemosensitivity by promoting BRCA1 expression and Smad3 phosphorylation. In vivo studies showed that TGF-ß1 knockdown significantly inhibited the growth of tumors, also by upregulating BRCA1 expression and Smad3 phosphorylation. CONCLUSION: Taken together, our results suggest that TGF-ß1 knockdown inhibits tumor growth and increases chemosensitivity by promotion of BRCA1/Smad3 signaling.

Investigation into the correlations of expressions of Cav-3 and Smad3 with pathogenesis and prognosis of viral myocarditis.

OBJECTIVE: To investigate the correlations of expressions of Caveolae-3 (Cav-3) and sma and mad homologue (Smad3) with the pathogenesis and prognosis of viral myocarditis (VMC). MATERIALS AND METHODS: VMC animal models were prepared and divided into the control group, the virus group and the Shenmai group. We detected the levels of creatine kinase isoenzyme (CK-MB) in the serum that was associated with the myocardial injuries, investigated the pathological features of VMC in BALB/C mice via hematoxylin-eosin (HE) staining, measured the mRNA expressions of Cav-3 and Smad3 via Real-time polymerase chain reaction (RT-PCR) and determined the protein expressions of Cav-3 and Smad3 through Western blotting method. RESULTS: The expressions of CK-MB in the virus group and Shenmai group were significantly higher than those in the control group; in comparison with the virus group, obvious improvement was identified in the pathologic condition of the Shenmai group; also, there was a statistically significant difference in comparison of the pathologic scores of BALB/C mice between the Shenmai group and the virus group. The mRNA expressions of Cav-3 and Smad3 in the virus group and Shenmai group were significantly higher than those in the control group, and the differences had statistical significance; however, higher mRNA expressions were identified in the virus group. Besides, protein expressions of Cav-3 and Smad3 in the virus group and Shenmai group were remarkably higher than those in the control group with statistically significant differences, but those in the virus group were much higher. CONCLUSIONS: Cav-3 and Smad3 may be involved in the occurrence and development of VMC, which provides some theoretical evidence for further research into the pathogenesis of VMC and the development of clinical drugs for treatment of VMC.

Granulosa Cell Tumors: Novel Predictors of Recurrence in Early-stage Patients.

Granulosa cell tumors (GCTs) comprise 2% to 5% of ovarian neoplasms, with unpredictable patterns of recurrence. The HER family, GATA4, and SMAD3 genes are reportedly involved in GCT proliferation and apoptosis and may serve as new predictors of recurrence. The aim of the study was to evaluate novel predictors of recurrence in GCT from a large single institution cohort. Patients diagnosed with GCTs (n=125) between 1975 and 2014 were identified. Clinicopathologic parameters were obtained and immunohistochemical evaluation was performed of calretinin, inhibin, HER2, CD56, SMAD3, and GATA4. Statistical analyses were conducted using Fisher exact test and Kaplan-Meier survival curves and Cox regression analysis. The median follow-up period was 120 months (range, 1-465 mo). Recurrence was noted in 12/125 (9.6%) patients. Kaplan-Meier analysis showed a shorter mean disease-free interval in whites versus blacks (P=0.001), stage III-IV versus stage I-II (P=0.0001), patients treated with surgery+chemotherapy versus surgery (P=0.0001), mitotic rate ≥4 (P=0.005), severe nuclear pleomorphism (P=0.013), high HER2 expression (P=0.001), high CD56 expression (P=0.001), and high SMAD3 expression (P=0.001). On Cox regression analysis, SMAD3 and type of treatment received were the only 2 independent prognostic factors for disease-free interval (P=0.03 and P=0.007, respectively). On subanalysis for early-stage (stage I) GCTs, the need for adjuvant chemotherapy and high expression of SMAD3 continued to be independent predictors of recurrence (HR=10.2, P=0.01 and HR=8.9, P=0.001, respectively).

ERBIN deficiency links STAT3 and TGF-ß pathway defects with atopy in humans.

Nonimmunological connective tissue phenotypes in humans are common among some congenital and acquired allergic diseases. Several of these congenital disorders have been associated with either increased TGF-ß activity or impaired STAT3 activation, suggesting that these pathways might intersect and that their disruption may contribute to atopy. In this study, we show that STAT3 negatively regulates TGF-ß signaling via ERBB2-interacting protein (ERBIN), a SMAD anchor for receptor activation and SMAD2/3 binding protein. Individuals with dominant-negative STAT3 mutations (STAT3mut ) or a loss-of-function mutation in ERBB2IP (ERBB2IPmut ) have evidence of deregulated TGF-ß signaling with increased regulatory T cells and total FOXP3 expression. These naturally occurring mutations, recapitulated in vitro, impair STAT3-ERBIN-SMAD2/3 complex formation and fail to constrain nuclear pSMAD2/3 in response to TGF-ß. In turn, cell-intrinsic deregulation of TGF-ß signaling is associated with increased functional IL-4Rα expression on naive lymphocytes and can induce expression and activation of the IL-4/IL-4Rα/GATA3 axis in vitro. These findings link increased TGF-ß pathway activation in ERBB2IPmut and STAT3mut patient lymphocytes with increased T helper type 2 cytokine expression and elevated IgE.

Heat shock transcription factor 1 protects against pressure overload-induced cardiac fibrosis via Smad3.

Fibrotic cardiac muscle exhibits high stiffness and low compliance which are major risk factors of heart failure. Although heat shock transcription factor 1 (HSF1) was identified as an intrinsic cardioprotective factor, the role that HSF1 plays in cardiac fibrosis remains unclear. Our study aims to investigate the role of HSF1 in pressure overload-induced cardiac fibrosis and the underlying mechanism. HSF1 phosphorylation was significantly downregulated in transverse aortic constriction (TAC)-treated mouse hearts and mechanically stretched cardiac fibroblasts (cFBs). HSF1 transgenic (TG) mice, HSF1 deficient heterozygote (KO) mice, and their wild-type littermates were subjected to sham or TAC surgery for 4 weeks. HSF1 overexpression significantly attenuated pressure overload-induced cardiac fibrosis and dysfunction. Conversely, HSF1 KO mice showed deteriorated fibrotic response and cardiac dysfunction upon TAC. Moreover, we uncovered that overexpression of HSF1 protected against fibrotic response of cFBs to pressure overload. Mechanistically, we observed that the phosphorylation and the nuclear distribution of the Smad family member 3 (Smad3) were significantly decreased in HSF1-overexpressing mouse hearts, while being greatly increased in HSF1 KO mouse hearts upon TAC, compared to the control hearts, respectively. Similar alteration of Smad3 phosphorylation and nuclear distribution were found in isolated mouse cardiac fibroblasts and mechanically stretched cFBs. Constitutively active Smad3 blocked the anti-fibrotic effect of HSF1 in cFBs. Furthermore, we found a direct binding of phosphorylated HSF1 and Smad3, which can be suppressed by mechanical stress. In conclusion, the present study demonstrated for the first time that HSF1 acts as a novel negative regulator of cardiac fibrosis by blocking Smad3 activation. KEY MESSAGES: HSF1 activity is decreased in fibrotic hearts. HSF1 overexpression attenuates pressure overload-induced cardiac fibrosis and dysfunction. Deficiency of HSF1 deteriorates fibrotic response and cardiac dysfunction upon TAC. HSF1 inhibits phosphorylation and nuclear distribution of Smad3 via direct binding to Smad3. Active Smad3 blocks the anti-fibrotic effect of HSF1.