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The cytokine TGFβ provides important information during embryonic development, adult tissue homeostasis, and regeneration. Alterations in the cellular response to TGFβ are involved in severe human diseases. To understand how cells encode the extracellular input and transmit its information to elicit appropriate responses, we acquired quantitative time-resolved measurements of pathway activation at the single-cell level. We established dynamic time warping to quantitatively compare signaling dynamics of thousands of individual cells and described heterogeneous single-cell responses by mathematical modeling. Our combined experimental and theoretical study revealed that the response to a given dose of TGFβ is determined cell specifically by the levels of defined signaling proteins. This heterogeneity in signaling protein expression leads to decomposition of cells into classes with qualitatively distinct signaling dynamics and phenotypic outcome. Negative feedback regulators promote heterogeneous signaling, as a SMAD7 knock-out specifically affected the signal duration in a subpopulation of cells. Taken together, we propose a quantitative framework that allows predicting and testing sources of cellular signaling heterogeneity., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

Elaborate regulatory networks of the Bone Morphogenetic Protein (BMP) pathways ensure precise signalling outcome during cell differentiation and tissue homeostasis. Here, we identified IRS4 as a novel regulator of BMP signal transduction and provide molecular insights how it integrates into the signalling pathway. We found that IRS4 interacts with the BMP receptor BMPRII and specifically targets Smad1 for proteasomal degradation consequently leading to repressed BMP/Smad signalling in C2C12 myoblasts while concomitantly activating the PI3K/Akt axis. IRS4 is present in human and primary mouse myoblasts, the expression increases during myogenic differentiation but is downregulated upon final commitment coinciding with Myogenin expression. Functionally, IRS4 promotes myogenesis in C2C12 cells, while IRS4 knockdown inhibits differentiation of myoblasts. We propose that IRS4 is particularly critical in the myoblast stage to serve as a molecular switch between BMP/Smad and Akt signalling and to thereby control cell commitment. These findings provide profound understanding of the role of BMP signalling in early myogenic differentiation and open new ways for targeting the BMP pathway in muscle regeneration.

Objective: Significant sex differences exist in cardiovascular diseases. Although an impact of gonadal hormones is presumed, it is largely unknown whether sexually dimorphic gene expression also plays a role and whether cells themselves show intrinsic sex differences., Methods: We performed whole genome expression analyses in human umbilical vein endothelial cells (HUVEC) from 20 male and 20 female donors and compared levels of gene transcription between the sexes. To further assess whether there is a sex-specific response to stress, we subjected male and female HUVEC to shear for 24 h and analysed changes in gene expression., Results: Genes indicative for greater immune responsiveness were stronger expressed in female compared to male HUVEC. There was a significant enrichment of 77 immune-related genes in female HUVEC. These increased transcriptional levels in female cells were verified for 20 genes by real-time RT-PCR. 6.7% of all mRNAs were regulated by shear stress. Female HUVEC showed a more pronounced transcriptional response to shear than did their male counterparts. In addition to quantitative differences, a number of genes were regulated in the opposite direction between the two sexes by shear stress. Functionally, female HUVEC showed a higher cell viability after serum starvation and an increased tube formation capacity compared to male cells., Conclusion: These findings underscore the importance for differentiation between male and female cells in cell culture experiments. This may apply not only to endothelial cells but might be generalized to other cell types as well. The observed sexual dimorphisms in gene expression in endothelial cells may contribute to sex differences between males and females in endothelial function., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

GDF8, or myostatin, is a member of the TGF-β superfamily of secreted polypeptide growth factors. GDF8 is a potent negative regulator of myogenesis both in vivo and in vitro. We found that GDF8 signaling was inhibited by the small molecule ATP competitive inhibitors dorsomorphin and LDN-193189. These compounds were previously shown to be potent inhibitors of BMP signaling by binding to the BMP type I receptors ALK1/2/3/6. We present the crystal structure of the type II receptor ActRIIA with dorsomorphin and demonstrate that dorsomorphin or LDN-193189 target GDF8 induced Smad2/3 signaling and repression of myogenic transcription factors. As a result, both inhibitors rescued myogenesis in myoblasts treated with GDF8. As revealed by quantitative live cell microscopy, treatment with dorsomorphin or LDN-193189 promoted the contractile activity of myotubular networks in vitro. We therefore suggest these inhibitors as suitable tools to promote functional myogenesis., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Bone Morphogenetic Proteins (BMPs) are members of the transforming growth factor-β superfamily of secreted polypeptide growth factors and are important regulators in a multitude of cellular processes. To ensure the precise and balanced propagation of their pleiotropic signaling responses, BMPs and their corresponding signaling pathways are subject to tight control. A large variety of regulatory mechanisms throughout different biological levels combines into a complex network and provides the basis for physiological BMP function. This regulatory network not only includes biochemical factors but also mechanical cues. Both BMP signaling and mechanotransduction pathways are tightly interconnected and represent an elaborate signaling network active during development but also during organ homeostasis. Moreover, its dysregulation is associated with a number of human pathologies. A more detailed understanding of this crosstalk in respect to molecular interactions will be indispensable in the future, in particular to understand BMP-related diseases as well as with regard to an efficient clinical application of BMP ligands., (© 2013 International Union of Biochemistry and Molecular Biology, Inc.)

Ligands of the Transforming Growth Factor β superfamily like Transforming Growth Factor β and Bone Morphogenetic Proteins govern developmental processes and regulate adult homeostasis by controlling cellular proliferation, survival, differentiation and migration. Aberrant signalling activity is associated with human disorders such as cancer, cardiovascular, musculoskeletal, or fibrotic disease. Upon binding to specific sets of cognate cell surface receptors, family members induce highly similar pathways which include canonical SMAD dependent signalling as well as pathways without direct involvement of SMAD proteins, which activate signalling molecules like mitogen-activated protein kinases or small GTPases. The diverse ligand functionalities are achieved through regulation and modulation of the pathways at all levels, resulting in a highly quantitative and context sensitive signal integration reflecting the cellular state and background. Strategies to target Transforming Growth Factor β or Bone Morphogenetic Protein pathways have been developed on the basis of our current understanding and have proven a highly beneficial potential., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Transforming growth factor-β (TGF-β) ligands and bone morphogenetic proteins (BMPs) play myriad roles in many biological processes and diseases. Their pluripotent activities are subject to multiple levels of regulation, including receptor oligomerization, endocytosis, association with co-receptors, cellular scaffolds or membrane domains, as well as transcriptional control. In this review, we focus on TGF-β and BMP receptor homomeric and heteromeric complex formation and their modulation by ligand binding, which regulate signaling on a near-immediate timescale. We discuss the current structural, biochemical and biophysical evidence for the oligomerization of these receptors, and the potential roles of distinct oligomeric interactions in signaling., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Elastin production is characteristically turned off during the maturation of elastin-rich organs such as the aorta. MicroRNAs (miRNAs) are small regulatory RNAs that down-regulate target mRNAs by binding to miRNA regulatory elements (MREs) typically located in the 3' UTR. Here we show a striking up-regulation of miR-29 and miR-15 family miRNAs during murine aortic development with commensurate down-regulation of targets including elastin and other extracellular matrix (ECM) genes. There were a total of 14 MREs for miR-29 in the coding sequences (CDS) and 3' UTR of elastin, which was highly significant, and up to 22 miR-29 MREs were found in the CDS of multiple ECM genes including several collagens. This overrepresentation was conserved throughout mammalian evolution. Luciferase reporter assays showed synergistic effects of miR-29 and miR-15 family miRNAs on 3' UTR and coding-sequence elastin constructs. Our results demonstrate that multiple miR-29 and miR-15 family MREs are characteristic for some ECM genes and suggest that miR-29 and miR-15 family miRNAs are involved in the down-regulation of elastin in the adult aorta.

Mutations in the gene encoding transforming growth factor-beta receptor type II (TGFBR2) have been described in patients with Loeys-Dietz syndrome (LDS), Marfan syndrome type 2 (MFS2) and familial thoracic aortic aneurysms and dissections (TAAD). Here, we present a comprehensive and quantitative analysis of TGFBR2 expression, turnover and TGF-β-induced Smad and ERK signaling activity for nine mutations identified in patients with LDS, MFS2 and TAAD. The mutations had different effects on protein stability, internalization and signaling. A dominant-negative effect was demonstrated for mutations associated with LDS and MFS2. No mutation showed evidence of an immediate cell-autonomous paradoxical activation of TGF-β signaling. There were no cell biological differences between mutations described in patients with LDS and MFS2. By contrast, R460C, which has been found in familial TAAD but not in MFS2 or LDS, showed a less-severe dominant-negative effect and retained residual Smad phosphorylation and transcriptional activity. TAAD is characterized primarily by thoracic aortic aneurysms or dissections. By contrast, MFS2 is characterized by numerous skeletal abnormalities, and patients with LDS additionally can display craniofacial and other abnormalities. Therefore, our findings suggest that the balance between defects in Smad and ERK signaling might be an important determinant of phenotypic severity in disorders related to mutations in TGFBR2.

RNA interference (RNAi) has been shown to be suitable to inhibit viruses in experimental setups and is considered a promising antiviral strategy that is currently being tested in various clinical trials. The present study provides an approach to design siRNAs with high potency against a virus-specific target gene. In recent years, several outbreaks of aseptic meningitis caused by an echovirus 30 (EV-30) infection have been described. Based on an initial set of 30 in silico designed siRNAs, six siRNAs targeting the 3D RNA-dependent RNA-Polymerase (3D(Pol)) of EV-30 were selected. All but one of them showed high efficiency in both, reporter and virus assays. A second aim of the study was to re-investigate the relevance of the decay-accelerating factor (DAF, also known as CD55) as cellular entry receptor of EV-30 by means of RNAi, a question which had been under debate in previous studies. Knockdown of DAF inhibited drastically infection by EV-30 indicating that DAF plays an important role either as an attachment factor or as a receptor.

Objectives: Our purpose was to study the ultrastructural morphology of the microvasculature of human endometrial adenocarcinoma and to determine the effect of this malignancy on cell-to-cell communication between the components of the microvasculature and with the other tissue compartments of human endometrium. Methods. Multiple cases of human endometrial adenocarcinoma were studied and graded by light microscopy. Six cases of Grade I and six cases of Grade II were selected. Two blocks per case were studied ultrastructurally., Results: In contrast to our expectation that the ultrastructure of tumor vessels would suggest a great deal of proliferation and new vessel formation, we found that tumor vessels displayed a high degree of cellular differentiation, in the form of numerous and varied cell-to-cell contacts, and large amounts of protein production., Conclusions: The morphology of the microvasculature of endometrial adenocarcinoma suggests an active rather than passive role in tumor vessels., (Copyright 1999 Academic Press.)

The nuclear channel system (NCS), giant mitochondria and subnuclear glycogen form a triad of ultrastructural features observed in normal human endometrial epithelium in response to progestational steroids. Both the giant mitochondria and subnuclear glycogen have been described in endometrial adenocarcinoma, but the NCS has not. This article reports the development of the NCS in adenocarcinoma treated with medroxyprogesterone acetate. Previous studies suggest that the NCS in normal tissue is a response to the acyl group in the 17-beta position of the D-ring of some progestational steroids, such as medroxyprogesterone acetate. Medroxyprogesterone acetate was administered to 12 postmenopausal women with endometrial adenocarcinoma. Hysterectomies were performed 8 to 20 days after treatment. Pretreatment specimens were also obtained on 8 of the 12 patients. Using standard electron microscopy procedures, light microscopy on plastic semithin sections was first used to confirm the presence of tumor. Thin sections of malignant endometrium were prepared and evaluated ultrastructurally for progestational alterations. Abnormal giant mitochondria and subnuclear glycogen were found both before and after treatment. The third element of the triad, the NCS, was not observed in any of the available pretreatment biopsies, but was seen in three of the treated specimens. Thus it appears that the NCS is a response to the given progesterone therapy.

Objective: Our purpose was to determine the effect of hyperplasia on cell-to-cell communication in the microvasculature of the human endometrium., Study Design: Ten biopsy specimens of simple and complex hyperplasia of the human endometrium were investigated by electron microscopy., Results: The microvasculature of the hyperplastic endometrium shows morphologic disorder. The extracellular matrix is altered, and cell death is widespread. Intercellular contact is infrequent or absent., Conclusions: These results are in marked contrast to the ultrastructure of the normal microvasculature. The complex cell-to-cell interaction observed in normal tissue appears lost. This report, combined with others, indicates that cell-to-cell contact is sparse in hyperplastic endometrium.

In hyperplastic endometrial stroma, cell-to-cell contacts are infrequent. Those that are present are not well developed and may appear degenerative. Significant amounts of extracellular matrix further impede contact. The cells of hyperplastic endometrial stromal are, for the most part, poorly differentiated and both apoptosis and necrosis are common. Fibroblast-like cells make up most of the increased cellularity, with few endometrial granulocytes or other cells types.

Background: A rare malignant germ cell tumor of the ovary during pregnancy was detected by screening of maternal serum alpha-fetoprotein (MSAFP). Treatment of this uncommon tumor during pregnancy incorporated combination chemotherapy including etoposide., Case: An 18-year-old primiparous woman undergoing antenatal genetic screening was found to have an extremely elevated MSAFP of 477.8 IU/mL, or 12.46 multiples of the median. Oophorectomy and staging laparotomy at 20.5 weeks' gestation resulted in the diagnosis of mixed germ cell tumor of the ovary, with both endodermal sinus tumor and grade 3 immature teratoma. The patient received three courses of cis-platinum, etoposide, and bleomycin. Maternal serum AFP titers had returned to normal pregnancy levels by the start of the second course. A healthy female infant was delivered at 39 weeks' gestation following induction of labor for pregnancy-induced hypertension., Conclusion: Elevated MSAFP levels may be a presenting sign of malignant ovarian germ cell neoplasms. This report describes both surgical and chemotherapeutic treatment of a germ cell malignancy during pregnancy, with delivery at term. To date, major fetal toxicity from chemotherapy has not been identified.

Objective: Our objective was to assess the cell-to-cell contacts in hyperplastic human endometrium and to compare them with contacts in normal endometrium., Study Design: Hyperplastic endometrial samples (five with simple hyperplasia and five with complex hyperplasia) without atypia by light microscopy were studied with transmission electron microscopy. The cell-to-cell contacts, basal lamina, and epithelial-to-stromal compartment contacts were studied and compared with these same areas in normal endometrium., Results: Only infrequent gap junctions between epithelial cells were seen. Other epithelial contacts were also greatly reduced when compared with normal epithelium. Cell-to-cell contacts between the epithelial compartment and the stromal compartment were rare and degenerative in configuration. An increase in extracellular matrix and various alterations in the basal lamina appears to contribute to a decrease in cell-to-cell contacts., Conclusions: Compared with normal endometrium, hyperplastic endometrium demonstrated far fewer cell-to-cell contacts. The basal lamina and extracellular matrix were altered in such a fashion that communication between the stromal and epithelial compartments appeared to be impaired. Stromal and epithelial cells rarely were in contact. Those contacts present were less complex and the majority were degenerative. Hyperplasia without atypia demonstrated a much lower amount of intercellular communication than normal endometrium.

During a 2-year period, 293 consecutive conizations performed by residents in obstetrics and gynecology were reviewed. One hundred sixty-one patients underwent laser excision and 132 women were treated by cold knife conization. Average operating times for laser excision and knife conization were 31 and 33 minutes, respectively. Combined minor and major complication rates for laser and knife cones were 19 and 30%, respectively. Major complications including cervical stenosis and heavy bleeding were seen in 20% of knife conizations and 6% of laser excisions. Average blood loss for knife conization was 65 mL, and average blood loss for laser conization was 34 mL. Among those examined in follow-up with cervical cytology and colposcopy, no patient in the laser excision group showed evidence of persistent dysplasia, compared with one patient (3%) in the knife conization group. The reported data suggest no difference in operating time, length of anesthesia, or success of treatment between laser excision and knife conization performed by residents in training. Fewer complications were seen in the laser excision group.

In the early proliferative phase endometrial capillary basal lamina is loosely formed and discontinuous. It becomes more distinct in the late proliferative phase and develops whorled extensions that include pericytes in the early secretory to midsecretory phase. Throughout the cycle both endothelial cells and pericytes possess cytoplasmic processes that extend through discontinuities in the basal lamina and make contact with each other. These contacts may be simple or complex. They become more elaborate as the cycle progresses to the midsecretory phase. Endothelial cells show progressive but heterogeneous differentiation into the midsecretory phase, with individual cell hypertrophy. In the late-secretory phase the endometrial stroma and the basal lamina are characterized by widespread degeneration, and the cell-to-cell contacts sharply decrease. However, endothelial cells remain relatively intact.

Increasing differentiation in the human endometrium is associated with an apparent decrease in intercellular contacts between endometrial stromal cells

Eighteen (18) previously untreated patients with grade I (9) and II (9) endometrial adenocarcinoma provided endometrial samples for biochemical analysis of estrogen and progesterone receptors. The same samples were evaluated by histology and ultrastructure. Progesterone receptor negative samples (PR less than 1000 fmole/g tissue) showed more ultrastructural characteristics of malignancy and a wider range of changes than the progesterone positive specimens (PR greater than 1000 fmole/g tissue). These findings appeared to be independent of estrogen receptor status. No significant ultrastructural changes were seen when grades I and II were compared.

A patient developed a large perineal hernia and prolapse of a myocutaneous neovagina 13 months after total pelvic exenteration for recurrent squamous cell cancer of the cervix and gracilis myocutaneous vaginal reconstruction. The neovagina was removed and the pelvic floor defect was repaired with preserved human dura. Twelve months postoperatively, the patient remained asymptomatic. Current and potential uses for human dura allografts are discussed.

Reproducible measures of the ultrastructural changes occurring throughout the normal human menstrual cycle are useful for defining and differentiating between normal and pathological states. Glandular endometrial functionalis epithelium obtained from five menstrual cycle subphases (early and late proliferative; and early, middle and late secretory) has been quantitated and compared for changes in nuclear and nucleolar size. These analyses indicate that (a) nuclear area significantly increases in the late secretory subphase, perhaps a requirement of endometrial regeneration; (b) nucleolar area peaks during the early secretory subphase, a necessary prelude for the later increased secretory activity; and (c) nucleolar area declines in the middle and late secretory subphases, probably due to falling hormone blood levels late in the cycle. In the proliferative phase, the majority of cells containing large nuclei and nucleoli have a relatively undeveloped cytoplasm, while the cytoplasm of those in the secretory phase contains glycogen. These cells were termed Stage 1 and 2 cells, respectively. The late secretory subphase contains some degenerating mature epithelial cells with large nuclei and small nucleoli as well as differentiated and regenerating cells with larger nucleoli. These measures establish a baseline of the normal changes in nuclear and nucleolar size as they occur throughout the normal human menstrual cycle.

Two types of direct contact between epithelial and stromal cells in patients with vaginal adenosis are documented in electron micrographs. In the first type, epithelial cells send cytoplasmic extensions through the basal lamina to achieve, at times, complete fusion with fibroblasts in the stromal compartment. In the second type, mast cells act as intermediaries between the epithelial and stromal compartments.

Fifteen multifilament strings on Dalkon Shield IUDs removed from patients were evaluated with scanning electron microscopy. All fifteen showed deterioration of the sheath which encloses the smaller inner filaments. The greatest area of deterioration was at the attachment knot which is usually intrauterine. Deterioration of the sheath also occurs when the device is within the uterine wall or intraperitoneal. The risk of pelvic infection may be attributed to this deterioration of the sheath and contamination from that site.

Quantitative methods were used to determine ultrastructural area differences in nuclei and nucleoli of normal proliferative endometrium as well as grades 1, 2, and 3 adenocarcinomas. Nuclear area distribution curves differed significantly among all groups. No significant differences were observed between nucleolar area distribution curves of normal proliferative and grade 1 adenocarcinomas. However, significant differences were found among distribution curves of all other groups. The computer-assisted high resolution ultrastructural examination provides a sensitive means of evaluating differences in nuclear and nucleolar areas of cells in normal and cancerous states.

Medroxyprogesterone acetate is a steroid compound similar to progesterone in structure and function. Thirty women were given 150 mg. of medroxyprogesterone intramuscularly, and samples of the endometrium were examined by light and electron microscopy. The initial response of normal human endometrium was maturation and then inhibition. By 50 days after injection, the atrophic changes began to reverse, and by Day 90, after injection the tissue resembled normal proliferative endometrium. A previously undescribed nuclear inclusion body is present; however, its significance is unclear at this time. On the basis of ultrastructural changes, medroxyprogesterone appeared not to exert a significant influence on the endometrium past 90 days at this dosage.

Patients with adequate colposcopic examinations and dysplasia on the endocervical curettage specimen usually undergo further diagnostic evaluation. This study evaluated 712 patients and found the frequency of positive endocervical curettage specimens to be 17.6%. Cervical conization or hysterectomy specimens were examined in 66 cases with a positive endocervical curettage. Of these, 69.7% had histologic evidence of discontinuous dysplasia in the endocervical canal. The endocervical curettage specimens were grouped by the percentage of endocervical epithelium that was dysplastic: group 1, less than 10%; group 2, 10% to 50%; group 3, greater than 50%. Confirmed histologic endocervical involvement was 80%, 56%, and 60%, respectively. The involvement of the endocervical canal also increased with increasing grade of cervical intraepithelial neoplasia. Of the 66 cases examined with positive endocervical curettage specimens, 62.1% had grade 3 cervical intraepithelial neoplasia on the ectocervix. Seven patients had positive endocervical margins, with six of these occurring in grade 3 cervical intraepithelial neoplasia lesions. Two unsuspected cases of cervical malignancy were identified by endocervical curettage.

Under the influence of progesterone the unique intranuclear structure known as the nucleolar canalicular structure (NCS) develops in the human endometrium. This organelle was not described previously outside the uterus. In this report the cases of two patients in whom the NCS appeared in extrauterine endometriosis are presented.

A study of the Groshong catheter in a gynecologic oncology population is presented, describing the catheter's ease of insertion, and patient acceptance and compliance in catheter care. From December 1985 through November 1987, 72 Groshong catheters were inserted in 67 patients. The Groshong differs from conventional Hickman-type catheters in design and maintenance. Thirty-two of 72 catheters (44%) were inserted under local anesthesia. The remaining catheters were inserted under general anesthesia at the time of major gynecologic procedures. None of the cases required fluoroscopy. The insertion technique is less traumatic than conventional approaches because of a stainless steel tunneling device. The median duration of use was 191 days. There were no cases of pneumothorax or catheter occlusion. Catheter removal was required in four cases with skin infection unresponsive to local therapy and in two cases with bacteremia. One patient developed thrombosis of the right subclavian vein but was treated without catheter removal. One patient with a skin infection at the exit site accidentally dislodged her catheter. The overall complication rate was 11% (8 of 72 cases).

This study is designed to analyze the complications of extended-field radiotherapy for carcinoma of the uterine cervix uncomplicated by recent prior surgery. Forty-two patients with carcinoma of the uterine cervix and lymph node metastases established by unequivocally positive bipedal lymphangiograms were treated with extended-field radiotherapy. External beam radiation to extended pelvic portals was limited to 4500 cGy using the linear accelerator and approximately 6000 mg-hr brachytherapy. Nodal boosts up to 500 cGy were generally limited to fields measuring less than 50 cm2. Higher doses were administered in 12 patients because of poor tumor regression. Eleven of these 12 patients experienced severe complications, and only three achieved control of their tumor. The type of treatment complication appeared to be directly related to specific modifications of the initial treatment plan. Treatment failures occurred within and outside of treatment portals with equal frequency.