The direct impact of pegvisomant on osteoblast functions and bone development.

PURPOSE: Acromegaly is a chronic disease characterized by growth hormone (GH) hypersecretion, usually caused by a pituitary adenoma, resulting in elevated circulating levels of insulin-like growth factor type I (IGF-I). Pegvisomant (PEG), the GH-receptor (GHR) antagonist, is used in treating acromegaly to normalize IGF-I hypersecretion. Exposure to increased levels of GH and IGF-I can cause profound alterations in bone structure that are not completely reverted by treatment of GH hypersecretion. Indeed, there is evidence that drugs used for the treatment of acromegaly might induce direct effects on skeletal health regardless of biochemical control of acromegaly. METHODS: We investigated, for the first time, the effect of PEG on cell proliferation, differentiation, and mineralization in the osteoblast cell lines MC3T3-E1 and hFOB 1.19 and its potential impact on bone development in zebrafish larvae. RESULTS: We observed that PEG did not affect osteoblast proliferation, apoptosis, alkaline phosphatase (ALP) activity, and mineralization. After PEG treatment, the analysis of genes related to osteoblast differentiation showed no difference in Alp, Runx2, or Opg mRNA levels in MC3T3-E1 cells. GH significantly decreased cell apoptosis (- 30 ± 11%, p < 0.001) and increased STAT3 phosphorylation; these effects were suppressed by the addition of PEG in MC3T3-E1 cells. GH and PEG did not affect Igf-I, Igfbp2, and Igfbp4 mRNA levels in MC3T3-E1 cells. Finally, PEG did not affect bone development in zebrafish larvae at 5 days post-fertilization. CONCLUSION: This study provides a first evidence of the impact of PEG on osteoblast functions both in vitro and in vivo. These findings may have clinically relevant implications for the management of skeletal health in subjects with acromegaly.

Atmospheric-Pressure Plasma-Enhanced Spatial ALD of SiO2 Studied by Gas-Phase Infrared and Optical Emission Spectroscopy.

An atmospheric-pressure plasma-enhanced spatial atomic layer deposition (PE-s-ALD) process for SiO2 using bisdiethylaminosilane (BDEAS, SiH2[NEt2]2) and O2 plasma is reported along with an investigation of its underlying growth mechanism. Within the temperature range of 100-250 °C, the process demonstrates self-limiting growth with a growth per cycle (GPC) between 0.12 and 0.14 nm and SiO2 films exhibiting material properties on par with those reported for low-pressure PEALD. Gas-phase infrared spectroscopy on the reactant exhaust gases and optical emission spectroscopy (OES) on the plasma region are used to identify the species that are involved in the ALD process. Based on the identified species, we propose a reaction mechanism where BDEAS molecules adsorb on -OH surface sites through the exchange of one of the amine ligands upon desorption of diethylamine (DEA). The remaining amine ligand is removed through combustion reactions activated by the O2 plasma species leading to the release of H2O, CO2, and CO in addition to products such as N2O, NO2, and CH-containing species. These volatile species can undergo further gas-phase reactions in the plasma as indicated by the observation of OH*, CN*, and NH* excited fragments in OES. Furthermore, the infrared analysis of the precursor exhaust gas indicated the release of CO2 during precursor adsorption. Moreover, this analysis has allowed the quantification of the precursor depletion yielding values between 10 and 50% depending on the processing parameters. Besides providing insights into the chemistry of atmospheric-pressure PE-s-ALD of SiO2, our results demonstrate that infrared spectroscopy performed on exhaust gases is a valuable approach to quantify relevant process parameters, which can ultimately help evaluate and improve process performance.

Ducky mouse phenotype of epilepsy and ataxia is associated with mutations in the Cacna2d2 gene and decreased calcium channel current in cerebellar Purkinje cells.

The mouse mutant ducky, a model for absence epilepsy, is characterized by spike-wave seizures and ataxia. The ducky gene was mapped previously to distal mouse chromosome 9. High-resolution genetic and physical mapping has resulted in the identification of the Cacna2d2 gene encoding the alpha2delta2 voltage-dependent calcium channel subunit. Mutations in Cacna2d2 were found to underlie the ducky phenotype in the original ducky (du) strain and in a newly identified strain (du(2J)). Both mutations are predicted to result in loss of the full-length alpha2delta2 protein. Functional analysis shows that the alpha2delta2 subunit increases the maximum conductance of the alpha1A/beta4 channel combination when coexpressed in vitro in Xenopus oocytes. The Ca(2+) channel current in acutely dissociated du/du cerebellar Purkinje cells was reduced, with no change in single-channel conductance. In contrast, no effect on Ca(2+) channel current was seen in cerebellar granule cells, results consistent with the high level of expression of the Cacna2d2 gene in Purkinje, but not granule, neurons. Our observations document the first mammalian alpha2delta mutation and complete the association of each of the major classes of voltage-dependent Ca(2+) channel subunits with a phenotype of ataxia and epilepsy in the mouse.

Peptides and vasomotor mechanisms.

The multiple and diverse roles played by neuropeptide Y, vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide and other biologically active peptides in the cardiovascular system are considered. A model of the vascular neuroeffector junction is described, which illustrates the interactions of peptidergic and nonpeptidergic transmitters that are possible at pre- and postjunctional sites. The effects of peptides on specific endothelial receptors are also described, which highlights the ability of these agents to act as dual regulators of vascular tone at both adventitial and intimal surfaces, following local release from nerves, or from endothelial cells themselves. Changes in expression of vascular neuropeptides that occur during development and aging in some disease situations and following nerve lesion are discussed.

Overlapping expression of zebrafish T-brain-1 and eomesodermin during forebrain development.

T-box transcription factors are important determinants of embryonic cell fate and behaviour. Two T-box genes are expressed in the developing telencephalon of several vertebrate species, including amphibia, birds and mammals. Here we report the cloning of zebrafish T-brain-1 (tbr1) and eomesodermin (eom). As a prelude to genetic studies of neuro-ectodermal fate determination we studied their expression pattern during embryogenesis and early larval development. Eom is expressed in the presumptive telencephalon from around the 4-5 somite stage in bilaterally symmetric groups of cells; the number of positive cells increases dramatically with time and encompasses the entire dorsal telencephalon by the 22 somite stage. Tbr1 is expressed from the 18 somite stage in a subset of eom-expressing cells. By 24 hpf eom and tbr1 are expressed in largely overlapping domains in the dorsal telencephalon, tbr1 is expressed in postmitotic cells whereas eomes is also expressed in proliferative ventricular zone cells. Both genes are also found in a small domain of the diencephalon bordering the telencephalon. A detailed analysis of the expression of tbr1 and eom in the brain of 4 day old larvae shows that the two T-box genes are differentially expressed in various cell populations of the developing brain.

DLX-1, DLX-2, and DLX-5 expression define distinct stages of basal forebrain differentiation.

The homeobox genes in the Dlx family are required for differentiation of basal forebrain neurons and craniofacial morphogenesis. Herein, we studied the expression of Dlx-1, Dlx-2, and Dlx-5 RNA and protein in the mouse forebrain from embryonic day 10.5 (E10.5) to E12.5. We provide evidence that Dlx-2 is expressed before Dlx-1, which is expressed before Dlx-5. We also demonstrate that these genes are expressed in the same cells, which may explain why single mutants of the Dlx genes have mild phenotypes. The DLX proteins are localized primarily to the nucleus, although DLX-5 also can be found in the cytoplasm. During development, the fraction of Dlx-positive cells increases in the ventricular zone. Analysis of the distribution of DLX-1 and DLX-2 in M-phase cells suggests that these proteins are distributed symmetrically to daughter cells during mitosis. We propose that DLX-negative cells in the ventricular zone are specified progressively to become DLX-2-expressing cells during neurogenesis; as these cells differentiate, they go on to express DLX-1, DLX-5, and DLX-6. This process appears to be largely the same in all regions of the forebrain that express the Dlx genes. In the basal telencephalon, these DLX-positive cells differentiate into projection neurons of the striatum and pallidum as well as interneurons, some of which migrate to the cerebral cortex and the olfactory bulb.

Use of enhanced silver staining combined with electron microscopical immunolabelling to demonstrate the colocalization of neuropeptide Y and vasoactive intestinal polypeptide in cerebrovascular nerves.

The combination of immunolabelling at the electron microscope level and enhanced silver staining has been used to demonstrate the colocalization of neuropeptide Y and vasoactive intestinal polypeptide in perivascular nerves supplying cerebral arteries of the rat. This has been shown in control tissue, but it is easier to demonstrate after long-term sympathectomy since that leads to an enhancement of neuropeptide Y in vasoactive intestinal polypeptide-containing parasympathetic nerves supplying these vessels. Immunolabelling of the antigens for these peptides was performed sequentially with the biotin streptavidin diaminobenzidine method, and the end product to the first antiserum was gold-silver intensified before the visualization of the second antigen. Using this technique, it was shown that all the neuropeptide Y immunoreactivity present in the rat cerebral vessels after long-term sympathectomy with guanethidine was localized in vasoactive intestinal polypeptide-containing nerves. Furthermore, an immunohistochemical analysis of the parasympathetic pterygopalatine ganglia in guanethidine-treated rats showed an increase in the percentage of neurons displaying neuropeptide Y immunoreactivity. In order to clarify if the pterygopalatine ganglion was the origin of those neuropeptide Y/vasoactive intestinal polypeptide-immunoreactive cerebrovascular nerves, which had increased in number after sympathectomy, a fluorescent neuronal tracer (Fast Blue) was applied to the right middle cerebral artery of rats which had undergone guanethidine treatment for six weeks. Immunohistochemical analysis of the ipsilateral ganglion 72 h after application of the tracer revealed the presence of immunoreactivity to both these peptides in retrogradely labelled neurons. It is concluded that neuropeptide Y and vasoactive intestinal polypeptide are colocalized in perivascular parasympathetic nerves supplying the middle cerebral artery of the rat, which have their origin in the pterygopalatine ganglion. Furthermore, long-term sympathectomy with guanethidine leads to an increase in the expression of neuropeptide Y in these vasoactive intestinal polypeptide-immunoreactive neurons.

Long-term chemical sympathectomy leads to an increase of neuropeptide Y immunoreactivity in cerebrovascular nerves and iris of the developing rat.

Short-term (surgical) and long-term (chemical) sympathectomy have revealed the presence of a population of neuropeptide Y-like immunoreactive nerve fibres which do not degenerate in parallel with noradrenaline-containing nerves supplying cerebral vessels and the iris of the rat. Two days after bilateral removal of the superior and middle cervical ganglia of 7-week-old rats, noradrenaline-containing nerves could not be detected along any of the arteries of the rat circle of Willis or of the iris, but 18-32% of neuropeptide Y-like immunoreactive nerves remained. Long-term treatment (6 weeks) with guanethidine commencing in developing 1-week-old rats caused degeneration of the sympathetic neurons in cervical ganglia and disappearance of 5-hydroxydopamine-labelled nerves (that showed dense-cored vesicles at the electron microscope level) from rat cerebral vessels, but did not significantly change the density of neuropeptide Y-like immunoreactive axons on the vessels. Furthermore, whilst in control rats neuropeptide Y-like immunoreactivity was localized largely within 5-hydroxydopamine-labelled cerebrovascular nerves, after long-term sympathectomy with guanethidine, neuropeptide Y-like immunoreactivity was seen only in nerves lacking small dense-cored vesicles. A small number of catecholamine-containing nerves appeared along the internal carotid and anterior cerebral arteries after long-term sympathectomy; these may arise from neurons of central origin. These results suggest that as a consequence of long-term sympathectomy with guanethidine, compensatory changes occur, involving an increase in the expression of neuropeptide Y-like immunoreactivity in non-sympathetic axons in cerebrovascular nerves and iris of the rat. In contrast, the neuropeptide Y-like immunoreactive nerves in the dura mater appear to be entirely sympathetic, since none were present after short-term sympathectomy and none appeared after long-term sympathectomy.

Binding of [3H]-muscimol to GABAA sites in the guinea-pig urinary bladder: biochemical assay and autoradiography.

1. The specific binding of [3H]-muscimol, a gamma-aminobutyric acidA receptor (GABAA) agonist, to whole membranes of the guinea-pig urinary bladder was examined. In addition, the distribution of specific muscimol binding sites within the bladder was visualized by autoradiography. 2. It was demonstrated that in a frozen-thawed whole membrane preparation of the organ the specific binding of [3H]-muscimol is reproducible, reversible and saturable. 3. Saturable binding was of a single component with an equilibrium dissociation constant (Kd) of 12 nM and a maximal density (Bmax) of about 80 fmol mg-1 protein. 4. In displacement experiments with several model compounds, [3H]-muscimol binding sites showed the characteristics of a GABAA receptor site. 5. Autoradiographic experiments revealed uneven distribution of specifically bound [3H]-muscimol in the bladder. The density of binding sites was high in clusters within the smooth muscle layers of the bladder fundus and of the urethra, while the apex and the neck were not specifically labelled. 6. The present findings show that GABAA type receptor sites in the guinea-pig urinary bladder may be labelled by [3H]-muscimol in a specific and reproducible manner. Moreover, the localization of these binding sites is consistent with the presence of GABAA receptors in only a subpopulation of vesical ganglia.

Nerve fibres in the uterine artery increase in number in pregnant guinea-pigs.

Perivascular nerves of the uterine artery were studied in four-month-old pregnant and non-pregnant guinea-pigs. In pregnancy, the uterine artery hypertrophies (more than two-fold growth in diameter and length and 50% increase in wall thickness), but its density of innervation remains high, because of the growth of many new axons. The axons in a complete transverse section of the vessel increase by 40%. The nerve bundles grow by 100%. Small nerve bundles increase more than large bundles: nearly 70% of the nerve bundles contain 10 or fewer axons in pregnant guinea-pigs (37% in non-pregnant animals). The increase in axon number is accompanied by formation of new varicosities, hence presumably of new neuromuscular junctions.

An increase in the expression of neuropeptidergic vasodilator, but not vasoconstrictor, cerebrovascular nerves in aging rats.

Perivascular nerve fibres containing noradrenaline (NA), serotonin (5-HT), substance P (SP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and calcitonin gene-related peptide (CGRP) were localized in whole-mount stretch preparations of the arteries of the rat circle of Willis using fluorescence and immunohistochemical techniques. Changes in the pattern and density of these perivascular nerves were studied from birth to 27 months of age. All perivascular nerve types reached a peak density of innervation at 1 month of age. This was followed by a general fall in the density of fluorescent nerve fibres. However, with aging, there was a decrease in the expression of vasoconstrictor neurotransmitters (NA and 5-HT) in cerebrovascular nerves, whereas the expression of vasodilator neurotransmitter (VIP and CGRP) in perivascular nerve fibres supplying the rat cerebral arteries was strikingly increased in old age. The density of NPY- and SP-containing nerve fibres was not significantly altered in old age. These changes are discussed in relation to the increased incidence of cerebrovascular disorders in the elderly.

Dopamine-sensitive cAMP generating system in rat extracerebral arteries.

The effect of dopamine (DA) on the 3',5'-cyclic adenosine monophosphate (cAMP) generating system in rat extracerebral arteries was studied in order to identify DA receptors. The carotis interna and the basilar artery as well as the arteries of the circle of Willis were homogenized and centrifuged. This extracellular arterial preparation was used for studying the effect of DA, norepinephrine and of drugs interfering with alpha- and beta-adrenergic as well as with dopaminergic receptors on cAMP production. DA increased the concentration of cAMP. The DA-elicited increase of cAMP levels was inhibited by dopamine receptor blocking agents (phenothiazine derivatives and butyrophenones) but unaffected by alpha- or beta-adrenoceptor blocking drugs or by the ergot-like DA agonist bromocriptine, or by sulpiride. The findings appear to be indicative of the existence of only D1 receptors within the blood vessels studied.

Characterization of [3H]5-hydroxytryptamine uptake within rat cerebrovascular tree.

The in vitro uptake of tritiated serotonin ([3H]5HT) was studied in a preparation of rat extracerebral arteries. The uptake of [3H]5HT was time- and temperature-dependent and of high affinity; linear regression analysis gave a Km value of 6.48 X 10(-7) M for the specific uptake. Bilateral superior cervical ganglionectomy was without effect on [3H]5HT uptake while it significantly reduced the uptake of tritiated norepinephrine by the preparation of rat extracerebral arteries. The serotonergic neurotoxin 5,7-dihydroxytryptamine and lesions to both the medial and the dorsal raphe nuclei caused a marked loss of [3H]5HT uptake but did not change the uptake of tritiated norepinephrine. Competition studies with norepinephrine, desimipramine (a noradrenergic uptake blocker), nomifensine (a dopaminergic uptake blocker) and fluoxetine (a 5HT uptake blocker) confirmed the specificity of the [3H]5HT uptake mechanism. Histoautoradiographic studies showed the highest density of silver grains at the level of the adventitial-medial border of the basilar artery. Fluoxetine inhibited the accumulation of silver grains within the adventitial-medial border in the blood vessel studied. The present data further support the view that a neuronal serotonergic system may play a role in the control of blood flow in the cerebrovascular tree.

Increase of dopamine beta-hydroxylase immunoreactivity in non-noradrenergic nerves of rat cerebral arteries following long-term sympathectomy.

The expression of dopamine beta-hydroxylase (DBH) and tyrosine hydroxylase (TH) immunoreactivity (IR) after short-term (2 days) and long-term (3 weeks) sympathectomy was investigated in rat cerebral vessels, dura mater and pterygopalatine ganglion neurones (which are known to project to cerebral arteries) by immunohistochemistry at both the light and electron microscopical levels. TH-IR, like glyoxylic acid-induced fluorescence, was completely abolished by sympathectomy. By contrast, DBH-IR was localized in nerve fibres, lacking 5-hydroxydopamine (5-OHDA)-labelled vesicles, along cerebral vessels of long-term sympathectomized rats, but not in the dura mater, and in pterygopalatine ganglia, where the number of DBH-IR neurons increased from 27.87% to 54.11%. Since virtually all the pterygopalatine neurons displayed choline acetyltransferase (ChAT)-IR, both in control and sympathectomized rats, it is concluded that long-term sympathectomy caused an increase of the expression of DBH-IR in cholinergic neurones of the pterygopalatine ganglion, without these neurons producing or storing noradrenaline.

Effect of long-term treatment with acetyl-L-carnitine on structural changes of ageing rat brain.

The effects of long-term treatment (11 months) with acetyl-L-carnitine (75 mg/kg daily) on the morphology of brain and optic nerve was studied in 16 senescent (22-month-old) Wistar rats (nine untreated, seven treated). Five young rats (aged 3 months) were used for comparison. Senescence was found to cause a structural disorganization of cerebral cortex, hippocampus and cerebellar cortex, and a decrease in the volume densities of the pyramidal neurons of layers 2 and 5 of the prefrontal cortex. An impaired myelination of the pyramidal tract and of the optic nerve was also observed. Besides improving the structural organization of the cerebral areas under study, treatment with acetyl-L-carnitine increased the volume densities of pyramidal neurons of the prefrontal cortex layers under observation. It must be added that myelination of the pyramidal tract and optic nerve was found to be less impaired after acetyl-L-carnitine administration.

Oncogene-induced reactive oxygen species fuel hyperproliferation and DNA damage response activation.

Oncogene-induced reactive oxygen species (ROS) have been proposed to be signaling molecules that mediate proliferative cues. However, ROS may also cause DNA damage and proliferative arrest. How these apparently opposite roles can be reconciled, especially in the context of oncogene-induced cellular senescence, which is associated both with aberrant mitogenic signaling and DNA damage response (DDR)-mediated arrest, is unclear. Here, we show that ROS are indeed mitogenic signaling molecules that fuel oncogene-driven aberrant cell proliferation. However, by their very same ability to mediate cell hyperproliferation, ROS eventually cause DDR activation. We also show that oncogenic Ras-induced ROS are produced in a Rac1 and NADPH oxidase (Nox4)-dependent manner. In addition, we show that Ras-induced ROS can be detected and modulated in a living transparent animal: the zebrafish. Finally, in cancer we show that Nox4 is increased in both human tumors and a mouse model of pancreatic cancer and specific Nox4 small-molecule inhibitors act synergistically with existing chemotherapic agents.

Targeting oncogene expression to endothelial cells induces proliferation of the myelo-erythroid lineage by repressing the Notch pathway.

Human oncogenes involved in the development of hematological malignancies have been widely used to model experimental leukemia. However, models of myeloid leukemia rarely reproduce the human disease in full, due to genetic complexity or to difficulties in targeting leukemia initiating cells. Here, we used a zebrafish genetic model to induce the expression of oncogenic RAS in endothelial cells, including the hemogenic endothelium of the dorsal aorta that generates hematopoietic cells, and observed the development of a myelo-erythroid proliferative disorder. In larvae, the phenotype is characterized by disruption of the vascular system and prominent expansion of the caudal hematopoietic tissue. In few surviving juveniles, increased number of immature hematopoietic cells and arrest of myeloid maturation was found in kidney marrow. Peripheral blood showed increased erythroblasts and myeloid progenitors. We found that the abnormal phenotype is associated with a downregulation of the Notch pathway, whereas overexpressing an activated form of Notch together with the oncogene prevents the expansion of the myelo-erythroid compartment. This study identifies the downregulation of the Notch pathway following an oncogenic event in the hemogenic endothelium as an important step in the pathogenesis of myelo-erythroid disorders and describes a number of potential effectors of this transformation.

Age-related changes in the noradrenergic innervation of the coronary arteries in old rats: a fluorescent histochemical study.

Age-related changes in the density of the noradrenergic perivascular plexus supplying the coronary vessels in the rat were studied using the glyoxylic acid fluorescence technique. A marked decrease in the density of the noradrenergic innervation of large, medium and small size coronary arteries was observed in 25-month-old rats. In contrast, the fluorescent nerves supplying the coronary veins were not decreased in old animals. The present data are indicative of an age-related reduction of fluorescent noradrenergic nerves in the rat coronary arteries.

The peptidergic innervation of the guinea pig uterine artery in pregnancy.

The influence of pregnancy on the density and pattern of the peptidergic innervation of the guinea pig uterine artery was studied. Whole mount stretch preparations of the uterine artery from estrus and late pregnant guinea pigs were processed for the immunohistochemical demonstration of neuropeptide Y (NPY)-, vasoactive intestinal polypeptide (VIP)-, calcitonin gene-related peptide (CGRP)- and substance P (SP)- immunoreactive nerve fibres. In late pregnancy the density of NPY- and CGRP- containing nerve fibres was remarkably decreased, while that of VIP- and SP- immunoreactive nerves showed a moderate reduction. The meaning and the possible physiological relevance of the decreased density of peptide-immunoreactive nerves in the uterine artery in late pregnancy are discussed.