Myhre Syndrome Misdiagnosed as Marfan Syndrome: an Educational Presentation

Abstract A 32-month-old girl with patent ductus arteriosus, false tendon of left ventricle, mild pulmonary hypertension, and chronic cardiac insufficiency (cardiac function level I-II) was misdiagnosed with Marfan Syndrome and there was no improvement in her physical growth after operation for this disease. The preterm baby was finally diagnosed with Myhre Syndrome by clinical phenotypes and mutation of SMAD4 gene.

Myhre Syndrome Misdiagnosed as Marfan Syndrome: an Educational Presentation.

A 32-month-old girl with patent ductus arteriosus, false tendon of left ventricle, mild pulmonary hypertension, and chronic cardiac insufficiency (cardiac function level I-II) was misdiagnosed with Marfan Syndrome and there was no improvement in her physical growth after operation for this disease. The preterm baby was finally diagnosed with Myhre Syndrome by clinical phenotypes and mutation of SMAD4 gene.

Dynamic Transcriptional and Epigenetic Changes Drive Cellular Plasticity in the Liver.

BACKGROUND AND AIMS: Following liver injury, a fraction of hepatocytes adopt features of biliary epithelial cells (BECs) in a process known as biliary reprogramming. The aim of this study was to elucidate the molecular events accompanying this dramatic shift in cellular identity. APPROACH AND RESULTS: We applied the techniques of bulk RNA-sequencing (RNA-seq), single-cell RNA-seq, and assay for transposase-accessible chromatin with high-throughput sequencing to define the epigenetic and transcriptional changes associated with biliary reprogramming. In addition, we examined the role of TGF-ß signaling by profiling cells undergoing reprogramming in mice with hepatocyte-specific deletion in the downstream TGF-ß signaling component mothers against decapentaplegic homolog 4 (Smad4). Biliary reprogramming followed a stereotyped pattern of altered gene expression consisting of robust induction of biliary genes and weaker repression of hepatocyte genes. These changes in gene expression were accompanied by corresponding modifications at the chromatin level. Although some reprogrammed cells had molecular features of "fully differentiated" BECs, most lacked some biliary characteristics and retained some hepatocyte characteristics. Surprisingly, single-cell analysis of Smad4 mutant mice revealed a dramatic increase in reprogramming. CONCLUSION: Hepatocytes undergo widespread chromatin and transcriptional changes during biliary reprogramming, resulting in epigenetic and gene expression profiles that are similar to, but distinct from, native BECs. Reprogramming involves a progressive accumulation of biliary molecular features without discrete intermediates. Paradoxically, canonical TGF-ß signaling through Smad4 appears to constrain biliary reprogramming, indicating that TGF-ß can either promote or inhibit biliary differentiation depending on which downstream components of the pathway are engaged. This work has implications for the formation of BECs and bile ducts in the adult liver.

BMP-6 and SMAD4 gene expression is altered in cumulus cells from women with endometriosis-associated infertility.

INTRODUCTION: Oocyte competence and quality depend on communication between the oocyte and the cumulus and theca cells. In the preantral phase, the members of the transforming growth factor ß (TGF-ß) superfamily are responsible for this communication and play an important role in folliculogenesis. Members of the TGF-ß superfamily are related to endometriosis (overexpression in the ectopic endometrium); however, few studies have explored these proteins as influencing fertility in endometriosis. Considering endometriosis-related infertility and to better understand the role of the TGF-ß superfamily members in the antral phase in women with endometriosis, this research investigated the gene expression of the genes for ligands AMH, BMP-6, GDF-9, INHA, INHBB, and TGFß3; receptors AMHR2, BMPR2, and TGFßR3; and intracellular signalling: SMAD3 and SMAD4. MATERIAL AND METHODS: The gene expression of AMH, BMP-6, GDF-9, INHA, INHBB, TGFß3, AMHR2, BMPR2, TGFßR3, SMAD3, and SMAD4 in cumulus cells was investigated through quantitative real-time PCR in a case-control study including infertile women with and without peritoneal endometriosis undergoing in vitro fertilization. RESULTS: Age and outcomes of assisted reproduction were similar between the groups (P > .05). However, women with endometriosis showed reduced expression of BMP-6 and SMAD4 (P < .05) in cumulus cells compared with the control group, other genes did not present altered gene expression in women with endometriosis (P > .05). CONCLUSIONS: The reduced expression of BMP-6 and SMAD4 in women with peritoneal endometriosis compared with the control group indicates that granulosa (cumulus) cell function could be altered in these women.

Pancreatic ductal adenocarcinomas from Mexican patients present a distinct genomic mutational pattern.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers in humans, with less than 5% 5-year survival rate. PDAC is characterized by a small number of recurrent mutations, including KRAS, CDKN2A, TP53, and SMAD4 and a long "tail" of infrequent mutated genes. Most of the studies have been performed in US and European populations, so new studies are needed to describe the mutational landscape of these tumors in other cohorts. The present study analyzed the exome and transcriptome of four PDAC tumors from Mexican patients. We found a paucity of the previously described recurrent mutations, with mutations in only three genes (HERC2, CNTNAP2 and HMCN1) previously reported in PDAC with a frequency > 1%. In addition, we discovered several recurrent putative copy number aberrations in SKP2, BRAF, CSSF1R, FOXE1, JAK2 and MET genes and in genes previously reported as putative drivers in PDAC, including KRAS, SF3B1, BRAF, MYC and MET. Although a larger cohort is needed to validate these findings, our results could be pointing toward potential differences in contributing factors for PDAC in Latin-American populations.

Effect of bone morphogenetic protein 9 on osteoblast differentiation of cells grown on titanium with nanotopography.

Among bone morphogenetic proteins (BMPs), BMP-9 has been described as one with higher osteogenic potential. Here, we aimed at evaluating the effect of BMP-9 on the osteoblast differentiation of cells grown on titanium (Ti) with nanotopography, a well-known osseoinductive surface. MC3T3-E1 cells were grown either in absence or presence of BMP-9 (20 nM) on Ti with nanotopography (Ti-Nano) or machined Ti (Ti-Machined) for up to 21 days to evaluate the gene expression of RUNX2, osterix, osteocalcin, bone sialoprotein, SMAD6 and SMAD4, protein expression of SMAD4, ALP activity and extracellular matrix mineralization. As expected BMP-9 increased osteoblast differentiation irrespective of Ti surface topography; however, the cells grown on Ti-Nano were more responsible to BMP-9 compared with cells grown on Ti-machined. This could be, at least in part, due to the fact that Ti-Nano may act on both ways, by increasing the activation (SMAD4) and decreasing the inhibition (SMAD6) of the signaling pathway triggered by BMP-9, while Ti-Machined only decrease the inhibition (SMAD6) of this pathway. In conclusion, the combination of the osteogenic potential of BMP-9 with the osseoinductive capacity of Ti-Nano could be a promising strategy to favor the osseointegration of Ti implants.

Applicability of the Curaçao Criteria for the Diagnosis of Hereditary Hemorrhagic Telangiectasia in the Pediatric Population.

OBJECTIVE: To evaluate the accuracy of the clinical Curaçao criteria in the diagnosis of hereditary hemorrhagic telangiectasia (HHT) in children and adolescents. STUDY DESIGN: This was a retrospective, multicenter chart review of 673 patients evaluated between 2002 and 2016; 290 were eligible for the study. Genetic testing for a pathogenic mutation was considered the gold standard against which the clinical Curaçao criteria were compared. Patients were divided into 4 age categories: 0-5, 6-10, 11-15, and 16-21-years. Sensitivity and specificity were calculated for each age group, and for the overall population. RESULTS: Overall the Curaçao criteria had a sensitivity of 68% (95% CI 60%-76%) and a specificity of 98% (95% CI 91%-100%). Sensitivity was lowest in the 0- to 5-year group, and increased with advancing age. The Curaçao criteria had the highest sensitivity in the 16- to 21-year-olds. Specificity was 100% in all age groups except for the 11- to 15-year-olds. CONCLUSIONS: This study evaluated the use of the Curaçao criteria for the diagnosis of HHT in the pediatric population with a family history of HHT. In those between the age of 0 and 21 years who meet 1 criterion (unlikely HHT) or 2 criteria (possible HHT), genetic testing is preferred for diagnosis. The Curaçao criteria appear to reliably diagnose HHT in children and adolescents who meet 3 or 4 criteria (definite HHT).

Sex differences in the development of prolactinoma in mice overexpressing hCGß: role of TGFß1.

Female transgenic mice that overexpress the human chorionic gonadotrophin ß subunit (hCGß+) develop prolactinomas, whereas hCGß+ males do not. The high levels of circulating hCG induce massive luteinization in the ovary of hCGß+ females, and progesterone becomes the primary steroid hormone produced, but estradiol remains at physiological level. The involvement of high levels of progesterone in lactotroph proliferation is not clearly understood; hence, the pathogenesis of prolactinomas in hCGß+ females remains unclear. TGFß1 is an inhibitor of lactotroph function, and the reduced TGFß1 activity found in prolactinomas has been proposed to be involved in tumor development. The aim of the present work was to study the role of TGFß1 in the gender-specific development of prolactinomas in hCGß+ mice. We compared the expression of different components of the pituitary TGFß1 system in males and females in this model. We found reduced TGFß1 levels, reduced expression of TGFß1 target genes, TGFß1 receptors, Ltbp1, Smad4 and Smad7 in hCGß+ female pituitaries. However, no differences were found between the transgenic and wild-type male pituitaries. We postulate that decreased pituitary TGFß1 activity in hCGß+ females is involved in the development of prolactinomas. In fact, we demonstrated that an in vivo treatment carried out for increasing pituitary TGFß1 activity, was successful in reducing the prolactinoma development, and the hyperprolactinemia in hCGß+ females. Moreover, the stronger TGFß1 system found in males could protect them from excessive lactotroph proliferation. Sex differences in the regulation of the pituitary TGFß1 system could explain gender differences in the incidence of prolactinoma.

Temporal and spatial expression of Smads and their correlation with YAP1 expression in sheep.

The temporal and spatial patterns of Smad and Yes-associated protein 1 (YAP1) expression were investigated in skeletal muscle (gastrocnemius muscle and extensor digitorum longus) at different growth stages (2 days old, 2 and 6 months old) in Hu sheep. Smads were differentially expressed in sheep skeletal muscle, with high expression in the gastrocnemius muscle and lower expression in the extensor digitorum longus. Expression of Smad2, Smad3, and Smad4 at the 2-day-old stage was significantly higher than at other stages (P < 0.05). The expression of Smad7 in 2-day-old sheep was lower than in 6-month-old sheep, with the lowest levels at 2 months. Smad expression was higher in males than in females at the 2-day-old stage, and expression in 2- and 6-month-old males was lower than that in 2-day-old females. Smad3 expression was higher in the 2-day- and 2-month-old males than in the females. There was a positive correlation (P < 0.01) between YAP1 and Smad2 expression in gastrocnemius muscle at the 2-month-old stage. YAP1 and Smad4/7 expression were positively correlated (P < 0.01) in extensor digitorum longus at the 2-day-old stage. YAP1 expression was negatively correlated with Smad7 in the extensor digitorum longus at 6 months. A significant difference between Smad2 and Smad3 (P < 0.01) expression in muscle was observed, consistent with Smad3 and Smad4 expression, indicating that these inhibit transforming growth factor-ß signaling in the same way. There was a positive correlation (P < 0.01) between YAP1 and MSTN expression, suggesting that YAP1 participates in muscle growth in sheep.

Analysis of the spatiotemporal expression of major genes in the TGF-ß/Smad signaling pathway and correlation analysis using Hu sheep muscle tissue.

The mRNA expression levels of key genes (Smads, MSTN, and MyoG) in the TGF-ß/Smad signaling pathway in Hu sheep at different growth stages (2 days, 2 months, and 6 months of age) and in different skeletal muscles (longissimus dorsi muscle and soleus muscle) and different genders were detected; and correlation of the Smad family (Smad2, Smad3, Smad4, and Smad7), MSTN, MyoG expressions was analyzed in Hu sheep. The results showed that the expression of Smads was higher in the soleus muscle than in the longissimus dorsi muscle; the expressions of Smad2, Smad3, and Smad4 were significantly higher in 2-day-old sheep than in sheep belonging to the other age groups (P < 0.05); the expressions of Smad2, Smad4, and Smad7 were higher in rams than in 2-day-old ewes, but lower in rams than in 2-month-old and 6-month-old ewes; and the expression of Smad3 was higher in rams than in 2-day-old and 2-month-old ewes, but lower in rams than in 6-month-old ewes. In the 2 different muscle tissues, expression of Smad2 was significantly positively correlated (P < 0.01) with that of Smad3. The expression of Smad3 was significantly positively correlated (P < 0.01) with that of Smad4, which showed that the Smad family genes could have an inhibitory effect on the TGF-ß/Smad signaling pathway.

Smad4 loss promotes lung cancer formation but increases sensitivity to DNA topoisomerase inhibitors.

Non-small-cell lung cancer (NSCLC) is a common malignancy with a poor prognosis. Despite progress targeting oncogenic drivers, there are no therapies targeting tumor-suppressor loss. Smad4 is an established tumor suppressor in pancreatic and colon cancer; however, the consequences of Smad4 loss in lung cancer are largely unknown. We evaluated Smad4 expression in human NSCLC samples and examined Smad4 alterations in large NSCLC data sets and found that reduced Smad4 expression is common in human NSCLC and occurs through a variety of mechanisms, including mutation, homozygous deletion and heterozygous loss. We modeled Smad4 loss in lung cancer by deleting Smad4 in airway epithelial cells and found that Smad4 deletion both initiates and promotes lung tumor development. Interestingly, both Smad4(-/-) mouse tumors and human NSCLC samples with reduced Smad4 expression demonstrated increased DNA damage, whereas Smad4 knockdown in lung cancer cells reduced DNA repair and increased apoptosis after DNA damage. In addition, Smad4-deficient NSCLC cells demonstrated increased sensitivity to both chemotherapeutics that inhibit DNA topoisomerase and drugs that block double-strand DNA break repair by non-homologous end joining. In sum, these studies establish Smad4 as a lung tumor suppressor and suggest that the defective DNA repair phenotype of Smad4-deficient tumors can be exploited by specific therapeutic strategies.

Smad4 loss in esophageal adenocarcinoma is associated with an increased propensity for disease recurrence and poor survival.

Previously regarded as a rare neoplasm, the incidence of esophageal adenocarcinoma has risen rapidly in recent decades. It is often discovered late in the disease process and has a dismal prognosis. Current prognostic markers including clinical, radiographic, and histopathologic findings have limited utility and do not consider the biology of this deadly disease. Genome-wide analyses have identified SMAD4 inactivation in a subset of tumors. Although Smad4 has been extensively studied in other gastrointestinal malignancies, its role in esophageal adenocarcinoma remains to be defined. Herein, we show, in a large cohort of esophageal adenocarcinomas, Smad4 loss by immunohistochemistry in 21 of 205 (10%) tumors and that Smad4 loss correlated with increased postoperative recurrence (P=0.040). Further, patients whose tumors lacked Smad4 had shorter time to recurrence (TTR) (P=0.007) and poor overall survival (OS) (P=0.011). The median TTR and OS of patients with Smad4-negative tumors was 13 and 16 months, respectively, as compared with 23 and 22 months, respectively, among patients with Smad4-positive tumors. In multivariate analyses, Smad4 loss was a prognostic factor for both TTR and OS, independent of histologic grade, lymphovascular invasion, perineural invasion, tumor stage, and lymph node status. Considering Smad4 loss correlated with postoperative locoregional and/or distant metastases, Smad4 was also assessed in a separate cohort of 5 locoregional recurrences and 43 metastatic esophageal adenocarcinomas. In contrast to primary tumors, a higher prevalence of Smad4 loss was observed in metastatic disease (44% vs. 10%). In summary, loss of Smad4 protein expression is an independent prognostic factor for TTR and OS that correlates with increased propensity for disease recurrence and poor survival in patients with esophageal adenocarcinoma after surgical resection.

Expression of Nodal, Cripto, SMAD3, phosphorylated SMAD3, and SMAD4 in the proliferative endometrium of women with endometriosis.

BACKGROUND: Nodal is a growth factor of the transforming growth factor ß superfamily that is expressed in high turnover tissues, such as the human endometrium, and in several malignancies. The effects of Nodal are modulated by the coreceptor Cripto and mediated by SMAD proteins. This study evaluated the gene and protein expression of Nodal, Cripto, total and phosphorylated (p) SMAD3, and SMAD4 in the proliferative endometrium of women with and without endometriosis. METHOD: Total RNA was isolated and complementary DNA synthesized from eutopic endometrium of women with (n = 15) and without (n = 12) endometriosis, followed by quantitative real-time polymerase chain reaction (PCR) to evaluate the gene expression of Nodal, Cripto, SMAD3, and SMAD4. Western blot was used to evaluate the protein levels of Nodal and Cripto, and immunohistochemistry was performed to localize SMAD3, pSMAD3, and SMAD4. RESULTS: Although Nodal expression was unchanged in women with endometriosis, real-time PCR indicated lower gene expression of Cripto (fold change 0.27, P < .05) in the endometriosis group. This difference, however, was not maintained at protein expression level as assessed by Western blot. The immunostaining of total SMAD3 was reduced in the endometriosis group (P < .01), but the localization of pSMAD3 and the nuclear staining of SMAD4 were unchanged. CONCLUSION: These findings suggest that the Nodal signaling pathway has subtle changes in the endometrium of women with endometriosis, but this imbalance may not cause functional damage as it seems not to affect the nuclear expression of SMAD4.

SMAD4 and TS expression might predict the risk of recurrence after resection of colorectal liver metastases.

PURPOSE: Colorectal liver metastases (CLM) have significant molecular heterogeneity, which contributes to the risk of recurrence following surgery. Most of the traditional scores intended to predict recurrence is based on clinicopathological variables and it is unclear whether incorporating molecular biomarkers might improve our assessment of the risk of recurrence. Our aim was to determine if molecular biomarkers might be associated with the risk of recurrence after surgery of CLM. PATIENTS AND METHODS: A total of 121 patients diagnosed with colorectal cancer (CRC) with resected liver metastases were included. The role of several clinicopathological variables to predict patient's outcome after resection of liver metastases was analyzed. Eighteen genes related to CRC pathogenesis were also included in the analyses. Univariate and multivariate stepwise Cox regression analyses were performed to identify factors associated with recurrence and the risk of death. RESULTS: Eight prognostic factors for progression-free survival and nine factors for overall survival were identified in the univariate analyses. After adjusting for other risk factors, only the expression of two molecular factors was associated with the risk of recurrence: TS (HR 0.631, 95 % CI 0.422-0.944) and SMAD4 (HR 1.680, 95 % CI 1.047-2.695). None of the variables was significantly associated with the risk of death in the multivariate analyses. CONCLUSIONS: The prognostic significance of most traditional clinicopathological variables might be insufficient to define patients at risk for recurrence after liver metastases resection. Molecular biomarkers might improve the identification of patients with higher risk of recurrence.

Oocyte-derived Smad4 is not required for development of the oocyte or the preimplantation embryo.

UNLABELLED: The generation of a competent egg requires complex molecular interactions between the oocyte and the ovary, and transforming growth factor ß (TGF-ß) is a major signaling pathway. Smad4 is a central regulator of the TGF-ß signaling pathway as it mediates gene expression triggered by activation of TGF-ß receptors. Deletion of Smad4 in granulosa cells disrupts follicle development; however, the role of Smad4 in the oocyte has not been confirmed. Furthermore, the role of Smad4 in embryo development has not been confirmed because previous studies of Smad4(del/del) embryos were generated from heterozygous parents, and thus it is possible that maternal transcripts rescue development before embryonic day 6.5 (E6.5) when Smad4(del/del) embryos die. To determine the role of TGF-ß signaling in oocyte and embryo development, mice with oocyte-specific deletion of Smad4 were studied. Fertility was evaluated in Mutant (Smad4(F/F):ZP3Cre) and CONTROL (Smad4(F/F)) females mated continuously with control males during a 6-month period. Surprisingly, Mutant females were fertile with the same litter size (Mutants, 9.23 ± 0.4; CONTROLs, 9.42 ± 0.4) and interlitter period as CONTROLs. Ovulation rate induced using a superovulation regime did not differ between CONTROLs and Mutants at both 6 weeks and 6 months. Embryo development was assessed at E6.5 using CONTROL and Mutant females mated with heterozygous males. Development of Smad4(del/del) embryos at E6.5 was retarded consistent with previous studies of embryos generated from heterozygous parents indicating that there is no rescue of preimplantation development by maternal transcripts. The numbers of implanted embryos at 6.5 dpc also did not differ ( CONTROL: 9.1 ± 0.4; Mutant: 7.0 ± 0.9). However, only 26.3% of E6.5 embryos carried by Mutant females were Smad4(del/del) compared with the expected ratio of 50%. Since litter size was not decreased, this indicates that either the number of Smad4(del) sperm fertilizing the oocytes is reduced or implantation of Smad4(del/del) embryos is suboptimal. In summary, we have shown that Smad4 in the oocyte, and thus TGF-ß signaling, is not required for oocyte or follicle development, ovulation, fertilization, preimplantation development, or implantation.

Gene expression differences in primary colorectal tumors and matched liver metastases: chemotherapy related or tumoral heterogeneity?

BACKGROUND: Treatment of metastatic colorectal cancer (mCRC) is generally based on genetic testing performed in primary tumor biopsies, but whether the genomic status of primary tumors is identical to that of metastases is not well known. We compared the gene expression profiles of formalin-fixed paraffin-embedded (FFPE) biopsies of colorectal primary tumors and matched liver metastases. PATIENTS AND METHODS: We compared the expression of 18 genes in FFPE CRC tumors and their matched liver metastases from 32 patients. The expression of each gene in CRC primary tumors and their matched liver metastases was tested using Student's t test for paired samples. Pairwise correlations of each gene in the primary tumors and matched liver metastases were evaluated by Pearson's correlation coefficient. RESULTS: The expression of six genes was significantly different in primary tumors compared with their matched liver metastases [CXCR4 (p < 0.001), THBS1 (p = 0.007), MMP 9 (p = 0.048), GST Pi (p = 0.050), TYMP (p = 0.042) and DPYD (p < 0.001)]. For the remaining genes, where no significant differences were observed, only SMAD4 (r s = 0.447, p = 0.010), ERCC1 (r s = 0.423, p = 0.016) and VEGF A (r s = 0.453, p = 0.009) showed significant correlation in expression between the two tissues. Therefore, we only detected similar gene expression levels between the tumor and the metastases in these three markers. CONCLUSIONS: We only found similar gene expression levels between the tumor and the metastases in three genes (SMAD4, ERCC1, and VEGF A). However, our study could not assess whether the differences in gene expression were secondary to tumoral heterogeneity or to molecular changes induced by previous chemotherapy.

Effect of BMPRIB gene silencing by siRNA on apoptosis and steroidogenesis of porcine granulosa cells.

Bone morphogenetic proteins (BMPs) are the key factors in maintaining cell growth and differentiation in ovaries. BMPs initiate signaling by assembling BMP receptors and activating Smads, which in turn alter the expression of target genes. However, little is known about the effect of the deletion of the Bone morphogenetic protein receptor type IB (BMPRIB) on porcine granulosa cell (GCs). The objective of this study was to determine the effects of BMPRIB gene silencing, by small interfering RNA (siRNA), on the apoptosis and steroidogenesis of porcine GCs, and the expression of cell cycle-related and apoptosis-related genes. Results indicate that the BMPRIB siRNA caused specific inhibition of BMPRIB mRNA expression after transfection. Knockdown of the BMPRIB gene significantly inhibited porcine GCs proliferation and estradiol production, while inducing apoptosis of porcine GCs. Additionally, the declined expression of the BMPRIB gene changed the expressions of CylinD2, Cdk2, Bcl-2, and Cyp19a1. These findings provide an important role of BMPRIB in the regulation of apoptosis and steroidogenesis of porcine GCs.

Age-dependent expression of Pten and Smad4 genes in the urogenital system of Wistar rats.

PURPOSE: To analyze Pten and Smad4 gene expression in the urogenital system of Wistar rats in differents ages. METHODS: Pten and Smad4 mRNA expression was assessed in the bladder, ventral prostate, testis, ovaries, and uterus by real-time PCR. Statistical analysis using the ANOVA (p<0.05). RESULTS: Pten levels showed a progressive age-dependent increase in the bladder (male and female) and prostate and were elevated in the ovaries of the middle-aged. In the uterus, no statistically significant differences were observed; in the testis, increased and decreased levels were seen in young adult and middle-aged rats, respectively. Smad4 expression was downregulated in the ovaries of the pubertal group but increased in the middle age group. In the uterus, Smad4 expression in the oldest group was higher than the others groups. In the testis, Smad4 expression steadily declined with age; in the prostate, it was higher in middle-aged rats than in younger rats. A similar trend was observed in the bladder of male and female middle-aged rats, compared with the pubertal group. CONCLUSION: The changes in phosphatase tensin homologue and Smad4 mRNA expression in Wistar rats appear to be associated with hormonal modifications in puberty and may be related to early follicular and testicular development.

Loss of SMAD4 protein expression is associated with high tumor grade and poor prognosis in disseminated appendiceal mucinous neoplasms.

The distinction between low-grade and high-grade disseminated appendiceal mucinous neoplasms is of critical importance in assessing prognosis and guiding patient therapy. SMAD4 encodes a protein that is a central component of the TGFß signal transduction pathway, and loss of SMAD4 expression has been associated with poor prognosis in carcinomas of the gastrointestinal tract. We reviewed the clinicopathologic and molecular features of 109 disseminated appendiceal mucinous neoplasms identified over an 8-year period at our institution in an attempt to: (1) correlate SMAD4 immunohistochemical expression with tumor grade; and (2) assess the prognostic significance of SMAD4 expression in predicting overall survival. Compared with tumors demonstrating preserved SMAD4 expression, tumors with loss of SMAD4 expression more frequently exhibited high cytologic grade (85% vs. 50%, P=0.035), high cellularity (100% vs. 45%, P<0.001), and destructive invasion (100% vs. 55%, P=0.001). SMAD4 expression significantly correlated with overall tumor grade (P=0.003): all 13 tumors with loss of SMAD4 expression were high grade, whereas all 42 low-grade tumors displayed preserved SMAD4 expression. A significantly higher proportion of tumors with loss of SMAD4 immunohistochemical expression demonstrated loss of heterozygosity at chromosome 18q (38%) compared with tumors with preserved SMAD4 expression (11%) (P=0.049), suggesting that loss of SMAD4 expression is due to genomic deletion in a high proportion of cases. Patients with SMAD4-negative tumors had significantly worse overall survival in comparison with patients with preserved SMAD4 expression (log rank P=0.023). However, our multivariable analysis found that SMAD4 expression was not independent of overall tumor grade in predicting overall survival. Our results indicate that loss of SMAD4 immunohistochemical expression is associated with loss of heterozygosity at chromosome 18q and is always associated with aggressive histologic features in disseminated appendiceal mucinous neoplasms. SMAD4 immunohistochemistry may be a useful ancillary study in select cases of disseminated appendiceal neoplasia, in which the distinction between low-grade and high-grade tumors is difficult.

The Ang-(1-7)/Mas-1 axis attenuates the expression and signalling of TGF-ß1 induced by AngII in mouse skeletal muscle.

AngII (angiotensin II) induces pathological conditions such as fibrosis in skeletal muscle. In this process, AngII increases ROS (reactive oxygen species) and induces a biphasic phosphorylation of p38 MAPK (mitogen-activated protein kinase). In addition, AngII stimulates the expression and production of TGF (transforming growth factor)-ß1 via a mechanism dependent on ROS production mediated by NADPH oxidase (NOX) and p38 MAPK activation. In the present study, we investigated whether Ang-(1-7) [angiotensin-(1-7)], through the Mas-1 receptor, can counteract the signalling induced by AngII in mouse skeletal muscle and cause a decrease in the expression and further activity of TGF-ß1 in skeletal muscle cells. Our results show that Ang-(1-7) decreased the expression of TGF-ß1 induced by AngII in a dose-dependent manner. In addition, we observed that Ang-(1-7) prevented the increase in TGF-ß1 expression induced by AngII, ROS production dependent on NOX and the early phase of p38 MAPK phosphorylation. Interestingly, Ang-(1-7) also prevented the late phase of p38 MAPK phosphorylation, Smad-2 phosphorylation and Smad-4 nuclear translocation, an increase in transcriptional activity, as determined using the p3TP-lux reporter, and fibronectin levels, all of which are dependent on the TGF-ß1 levels induced by AngII. We also demonstrated that Ang-(1-7) prevented the increase in TGF-ß1, fibronectin and collagen content in the diaphragm of mice infused with AngII. All of these effects were reversed by the administration of A779, indicating the participation of Mas-1. In conclusion, our findings support the hypothesis that Ang-(1-7) decreases the expression and further biological activity of TGF-ß1 induced by AngII in vitro and in vivo.