Sex-Based Differences in Outcomes Following Peripheral Artery Revascularization: Insights From VOYAGER PAD.

Background Despite high female prevalence of peripheral artery disease (PAD), little is known about sex-based outcomes after lower extremity revascularization (LER) for symptomatic PAD. The effects of rivaroxaban according to sex following LER have not been fully reported. Methods and Results In VOYAGER PAD (Vascular Outcomes Study of ASA [acetylsalicylic acid] Along with Rivaroxaban in Endovascular or Surgical Limb Revascularization for Peripheral Artery Disease), low-dose rivaroxaban versus placebo on a background of aspirin reduced the composite primary efficacy outcome of cardiovascular and limb events in patients with PAD undergoing LER. Unplanned index limb revascularization was prespecified and prospectively ascertained. The primary safety outcome was Thrombolysis in Myocardial Infarction major bleeding. Analyses of outcomes and treatment effects by sex were performed using Cox proportional hazards models. Among 6564 randomly assigned patients followed for a median of 28 months, 1704 (26.0%) were women. Among patients administered placebo, women were at similar risk for the primary efficacy outcome (hazard ratio [HR], 0.90; [95% CI, 0.74-1.09]; P=0.29) as men, while female sex was associated with a trend toward higher risk of unplanned index limb revascularization (HR, 1.18; [95% CI, 1.00-1.40]; P=0.0499). Irrespective of sex, effects of rivaroxaban were consistent for the primary efficacy outcome (P-interaction=0.22), unplanned index limb revascularization (P-interaction=0.64), and bleeding (P-interaction=0.61). Women were more likely than men to discontinue study treatment (HR, 1.13; [95% CI, 1.03-1.25]; P=0.0099). Conclusions Among >1700 women with PAD undergoing LER, women and men were at similar risk for the primary outcome, but a trend for greater risk of unplanned index limb revascularization among women was observed. Effects of rivaroxaban were consistent by sex, though women more often discontinued treatment. Better understanding of sex-based outcomes and treatment adherence following LER is needed. Registration URL: http://clinicaltrials.gov; Unique identifier: NCT02504216

Genetic control of hypothalamo-pituitary axis development and function in mice.

Congenital dysfunction of the hypothalamic-pituitary (HP) axis occurs in approximately one birth per 2,200 and is associated with a broad range of common disease states including impaired growth (short stature), infertility, hypogonadism poor responses to stress and slow metabolism (Pescovitz and Eugster, 2004). Although, a number of genes have been linked to diseases of the HP axis, the genetic cause in many patients remains unknown. This thesis examines two aspects of HP axis development and function. The first aim was to identify Sox3 targets by examining gene expression differences between three mouse lines: Sox3-null (mice lacking Sox3; loss of function), Extra-Sox3 (mice overexpressing Sox3; gain of function) and wild-type, by genome wide profiling using the Illumina BeadChip microarray platform. The second aim was to characterize the downstream effects relative to HP development in a novel recessive dwarf mouse model with pituitary hypoplasia and growth hormone (GH) deficiency, generated by N-ethyl-Nnitrosourea (ENU) mutagenesis that produces a point mutation in the gene for the enzyme tryptophanyl-tRNA synthetase (WARS). The first project (project 1) examined Sox3, the causative gene associated with Xlinked hypopituitarism (XH), in wild-type and transgenic mice. SOX3 is a member of the SOX (SRY-related HMG box) gene family of transcription factors that is expressed in progenitor cells of the mouse embryonic central nervous system (CNS) including the developing and postnatal hypothalamus (Rizzoti et al., 2004). It is the only member of the SOXB1 subfamily positioned on the X chromosome (Collignon et al., 1996; Stevanovic, 2003). Appropriate dose- and time-dependent expression of Sox3 in the developing hypothalamus is required for normal neuroendocrine function, particularly related to growth and growth hormone (GH). Changes associated with a loss-of-function and/or gain-of-function of Sox3 may contribute to a better understanding of other important genes, currently not known, involved in XH and/or X-linked mental retardation. At this point, however, the mechanisms linking SOX3 to its direct targets and their interplay within other downstream signaling cascades regulating HP axis development remain unknown. In order to identify Sox3-dependent genes, in mice, I performed microarray analysis of RNA extracted from embryonic mouse heads at 10.5 days post coitum (dpc) and compared RNA from wild-type, loss-of-function (Sox3-null) and gain-of-function (Extra-Sox3) mice. Several emergent candidate genes were further tested by quantitative mRNA expression analysis (qPCR). One of these was Neurogenin-3 (Ngn3), which showed a 2.5- fold decrease (P<0.001) in expression by microarray in Sox3-null (n=6), compared with wild-type (WT; n=6) mice and 1.8-fold decrease (P<0.001) by qPCR between Sox3-null (n=6) and WT (n=6) mice. To evaluate the relationship between Ngn3 and Sox3 at a cellular level immunohistochemistry was performed on 10.5 dpc and 12.5 dpc brains. In WT mice at 10.5 dpc and 12.5 dpc Ngn3 and Sox3 expression overlapped in a subset of cells across the ventral-midline of the developing hypothalamus. In addition and in contrast to WT mice, in Sox3-null mice, there were few Ngn3 positive cells, localized to the arcuate hypothalamic nucleus. Neurogenin-3 (Ngn3) is a member of the Neurogenin gene family of proneural basic helix-loop-helix proteins. Although previous data show the importance of Ngn3 during pancreatic development, there is no information on the mechanisms and actions of Ngn3 or a relationship between NGN3 action and SOX3 during hypothalamic development. These results suggest Ngn3 is a downstream target of Sox3 that is contributing to appropriate development of the hypothalamic-pituitary axis. The second study (project 2) aimed to characterize and further examine a novel recessive ENU mouse mutant, called Tukku¹ , exhibiting HP axis dysfunction resulting in dwarfism, pituitary hypoplasia and GH deficiency. Adult Tukku mice are 30-40% smaller than their WT littermates. The primary focus was to characterize the dwarfism phenotype in relation to the somatotropic axis and to identify the causative gene. The mutation was identified as a leucine to proline substitution in tryptophanyl-tRNA synthetase (WARS), a member of the aminoacyl-tRNA synthetase (AARS) enzyme family that link amino acids to their specific tRNAs. For proper function of this enzyme the specific recognition of substrates is critical for the fidelity of protein synthesis. The Wars mutation is contained within the N-terminal WHEP domain, from residue 16-69, and likely causes the disruption of the alpha helical structure. The N-terminal WHEP domain has only been found in eukaryote Wars enzyme. Importantly, AARS have been linked to regulating the noncanonical activity of angiogenesis (Otani et al., 2002; Wakasugi, 2010; Wakasugi and Schimmel, 1999; Wakasugi et al., 2002b). Along with pituitary hypoplasia, Tukku mice show a significant reduction in pituitary GH and serum levels of IGF-1, suggesting the defect leading to pituitary hypoplasia involves brain regions implicated in growth of the anterior pituitary. The reduction in pituitary GH levels may also involve delivery of GHreleasing hormone (GHRH) to GH-secreting cells since preliminary data also indicate that WARS is expressed within blood vessels of the pituitary and hypothalamus. To assess this, quantitative mRNA expression analysis (qPCR) of GHRH and somatostatin (Sst) was performed. qPCR revealed a decrease in both GHRH and Sst (fold change >2) indicating that the defect is likely to be within the hypothalamic hypophysial vasculature that extends and makes a connection with the pituitary. To evaluate the relationship between Wars and pituitary vasculature, immunohistochemistry was performed on pituitaries at 8-weeks postnatal. Pituitary sections were co-stained with antibodies against platelet endothelial cell adhesion molecule (PECAM) + Wars or vascular-endothelial cadherin (VE-Cadherin; an endothelial specific, transmembrane protein, which clusters at adheren junctions where it promotes homotypic cell-cell adhesion) + Wars. Wars immunostaining was expressed within the endothelial cells of the pituitary vasculature, both in the anterior and posterior pituitary. Both PECAM and Wars appeared co-expressed within the vascular wall. VECadherin was expressed in vessels together with Wars. Overall, the data gathered from these projects highlight important insights into the identification of Ngn3 as a likely Sox3 target gene (project 1) and have identified a novel dwarf mouse model with a genetic determinant of HP axis function (project 2). These results have application to the study of HP axis development, to the study of vascular development during embryology and postnatally, and to possible avenues of genetic screen testing and development of new treatments related to GH deficiencies. ¹ Tukku, meaning ‘small’ in Kaurna Aboriginal language.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 201

The importance of scanning electron microscopy (sem) in taxonomy and morphology of Chironomidae (Diptera)

The paper reports on the value of scanning electron microscopy (SEM) in the taxonomy and morphology of Chironomidae. This method has been relatively rarely used in Chironomidae studies. Our studies suggest that the SEM method provides a lot of new information. For example, the plastron plate of the thoracic horn of Macropelopia nebulosa (Meigen) under light microscopy is visible as points, while under SEM we have found that it consists of a reticular structure with holes. By using SEM a more precise picture of the body structure of Chironomidae can be revealed. It allows researchers to explain inconsistencies in the existing descriptions of species. Another advantage of the SEM method is obtaining spatial images of the body and organs of Chironomidae. However, the SEM method also has some limitations. The main problem is dirt or debris (e.g. algae, mud, secretions, mucus, bacteria, etc.), which often settles on the external surface of structures, especially those which are uneven or covered with hair. The dirt should be removed after collection of chironomid material because if left in place it can become chemically fixed to various surfaces. It unnecessarily remains at the surface and final microscopic images may contain artifacts that obscure chironomid structures being investigated. In this way many details of the surface are thus unreadable. The results reported here indicate that SEM examination helps us to identify new morphological features and details that will facilitate the identification of species of Chironomidae and may help to clarify the function of various parts of the body. Fast development of electron microscope technique allows us to learn more about structure of different organisms

Celecoxib reduces glucocorticoids in vitro and in a mouse model with adrenocortical hyperplasia

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PKA regulatory subunit 1A inactivating mutation induces serotonin signaling in primary pigmented nodular adrenal disease

International audiencePrimary pigmented nodular adrenocortical disease (PPNAD) is a rare cause of ACTH-independent hypercortisolism. The disease is primarily caused by germline mutations of the protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene, which induces constitutive activation of PKA in adrenocortical cells. Hypercortisolism is thought to result from PKA hyperactivity, but PPNAD tissues exhibit features of neuroendocrine differentiation, which may lead to stimulation of steroidogenesis by abnormally expressed neurotransmitters. We hypothesized that serotonin (5-HT) may participate in the pathophysiology of PPNAD-associated hypercortisolism. We show that PPNAD tissues overexpress the 5-HT synthesizing enzyme tryptophan hydroxylase type 2 (Tph2) and the serotonin receptors types 4, 6, and 7, leading to formation of an illicit stimulatory serotonergic loop whose pharmacological inhibition in vitro decreases cortisol production. In the human PPNAD cell line CAR47, the PKA inhibitor H-89 decreases 5-HT4 and 5-HT7 receptor expression. Moreover, in the human adrenocortical cell line H295R, inhibition of PRKAR1A expression increases the expression of Tph2 and 5-HT4/6/7 receptors, an effect that is blocked by H-89. These findings show that the serotonergic process observed in PPNAD tissues results from PKA activation by PRKAR1A mutations. They also suggest that Tph inhibitors may represent efficient treatments of hypercortisolism in patients with PPNAD

Embryonic stem cell factor FOXD3 (Genesis) defects in gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms, believed to originate from the interstitial cells of Cajal (ICC), often caused by overexpression of tyrosine kinase receptors (TKR) KIT or PDGFRA. Here, we present evidence that the embryonic stem cell factor FOXD3, first identified as \u27Genesis\u27 and involved in both gastrointestinal and neural crest cell development, is implicated in GIST pathogenesis; its involvement is investigated both in vitro and in zebrafish and a mouse model of FOXD3 deficiency. Samples from a total of 58 patients with wild-type GISTs were used for molecular analyses, including Sanger sequencing, comparative genomic hybridization, and methylation analysis. Immunohistochemistry and western blot evaluation were used to assess FOXD3 expression. Additionally, we conducted in vitro functional studies in tissue samples and in transfected cells to confirm the pathogenicity of the identified genetic variants. Germline partially inactivating FOXD3 sequence variants (p.R54H and p.Ala88_Gly91del) were found in patients with isolated GISTs. Chromosome 1p loss was the most frequent chromosomal abnormality identified in tumors. In vitro experiments demonstrate the impairment of FOXD3 in the presence of those variants. Animal studies showed disruption of the GI neural network and changes in the number and distribution in the ICC. FOXD3 suppresses KIT expression in human cells; its inactivation led to an increase in ICC in zebrafish, as well as mice, providing evidence for a functional link between FOXD3 defects and KIT overexpression leading to GIST formation