Characterization of atherosclerotic plaques in blood vessels with low oxygenated blood and blood pressure (Pulmonary trunk): role of growth differentiation factor-15 (GDF-15).

BACKGROUND: Growth differentiation factor (GDF)-15 is linked to inflammation, cancer, and atherosclerosis. GDF-15 is expressed in most tissues but is extremely induced under pathological conditions. Elevated serum levels are suggested as a risk factor and a marker for cardiovascular diseases. However, the cellular sources and the effects of GDF-15 on the cardiovascular system have not been completely elucidated including progression, and morphology of atherosclerotic plaques. Thus, this work aimed to characterize the influence of GDF-15 deficiency on the morphology of atherosclerotic plaques in blood vessels with low-oxygen blood and low blood pressure as the pulmonary trunk (PT), in hypercholesterolemic ApoE-/- mice. METHODS: GDF-15-/- ApoE-/- mice were generated by crossbreeding of ApoE-/-- and GDF-15-/- mice. After feeding a cholesterol-enriched diet (CED) for 20 weeks, samples of the brachiocephalic trunk (BT) and PT were dissected and lumen stenosis (LS) was measured. Furthermore, changes in the cellularity of the PT, amounts of apoptosis-, autophagy-, inflammation- and proliferation-relevant proteins were immunohisto-morphometrically analyzed. Additionally, we examined an atherosclerotic plaque in a human post mortem sample of the pulmonary artery. RESULTS: After CED the body weight of GDF-15-/-ApoE-/- was 22.9% higher than ApoE-/-. Double knockout mice showed also an 35.3% increase of plasma triglyceride levels, whereas plasma cholesterol was similar in both genotypes. LS in the BT and PT of GDF-15-/-ApoE-/- mice was significantly reduced by 19.0% and by 6.7% compared to ApoE-/-. Comparing LS in PT and BT of the same genotype revealed a significant 38.8% (ApoE-/-) or 26.4% (GDF-15-/-ApoE-/-) lower LS in the PT. Immunohistomorphometry of atherosclerotic lesions in PT of GDF-15-/-ApoE-/- revealed significantly increased levels (39.8% and 7.3%) of CD68 + macrophages (MΦ) and α-actin + smooth muscle cells than in ApoE-/-. The density of TUNEL + , apoptotic cells was significantly (32.9%) higher in plaques of PT of GDF-15-/-ApoE-/- than in ApoE-/-. Analysis of atherosclerotic lesion of a human pulmonary artery showed sm-α-actin, CD68+, TUNEL+, Ki67+, and APG5L/ATG+ cells as observed in PT. COX-2+ and IL-6+ immunoreactivities were predominantly located in endothelial cells and subendothelial space. In BT and PT of GDF15-/-ApoE-/- mice the necrotic area was 10% and 6.5% lower than in ApoE-/-. In BT and PT of GDF15-/-ApoE-/- we found 40% and 57% less unstable plaques than ApoE-/- mice. CONCLUSIONS: Atherosclerotic lesions occur in both, BT and PT, however, the size is smaller in PT, possibly due to the effect of the low-oxygen blood and/or lower blood pressure. GDF-15 is involved in atherosclerotic processes in BT and PT, although different mechanisms (e.g. apoptosis) in these two vessels seem to exist.

Associations of temporal and energetic characteristics of behavior with depressive symptoms: A population-based longitudinal study within Strelau's Regulative Theory of Temperament.

BACKGROUND: Individual dispositions have previously been associated with increased risk for depressive symptoms. The direction of the association has been found to be sometimes reciprocal. We examined whether temperament traits are associated with depressive symptoms and whether depressive symptoms contribute to changes in temperament. METHODS: Participants (n=674-811) were from a population-based Young Finns Study. Temperament was assessed by a Finnish version of the Formal Characteristics of Behavior - Temperament Inventory. Depressive symptoms were assessed with modified BDI (mBDI) in 1997, 2001, 2007 and 2012, and BDI-II in 2012. RESULTS: Higher perseveration and emotional reactivity were associated with higher level of depressive symptoms, and higher endurance was associated with lower level of depressive symptoms in 2007 and 2012. These associations were independent of several potential confounders and baseline depressive symptoms. The results of cross-lagged structural equation modeling showed that the associations between temperament and depressive symptoms were reciprocal: briskness, endurance and activity decreased the risk for depressive symptoms while depressive symptoms decreased the level of these characteristics. Perseveration, emotional reactivity and depressive symptoms reinforced each other over time. LIMITATIONS: The depressive symptoms scales we used are not meant for measuring clinically diagnosed depression. The relationships between temperament traits and depressive symptoms were not strong enough to provide a clinical basis for guiding treatment. CONCLUSIONS: Lower perseveration, lower emotional reactivity and higher endurance seem to be health protective temperament characteristics that reduce the risk for depressive symptoms. The reciprocal associations between temperament and depressive symptoms imply mutual health protective and health declining effects. Clinical relevance of the study is that enhancing positive loops and self-concept, and supporting individual stress management might be helpful in prevention of depressive symptoms.

F-spondin regulates neuronal survival through activation of disabled-1 in the chicken ciliary ganglion.

The extracellular membrane-associated protein F-spondin has been implicated in cell-matrix and cell-cell adhesion and plays an important role in axonal pathfinding. We report here that F-spondin is expressed in non-neuronal cells in the embryonic chicken ciliary ganglion (CG) and robustly promotes survival of cultured CG neurons. Using deletion constructs of F-spondin we found that the amino-terminal Reelin/Spondin domain cooperates with thrombospondin type 1 repeat (TSR) 6, a functional TGFß-activation domain. In ovo treatment with blocking antibodies raised against the Reelin/Spondin domain or the TSR-domains caused increased apoptosis of CG neurons during the phase of programmed cell death and loss of about 30% of the neurons compared to controls. The Reelin/Spondin domain receptor - APP and its downstream signalling molecule disabled-1 are expressed in CG neurons. F-spondin induced rapid phosphorylation of disabled-1. Moreover, both blocking the central APP domain and interference with disabled-1 signalling disrupted the survival promoting effect of F-spondin. Taken together, our data suggest that F-spondin can promote neuron survival by a mechanism involving the Reelin/Spondin and the TSR domains.

Molecular cloning, tissue expression, and partial characterization of the major fish CNS myelin protein 36k.

A full-length cDNA clone encoding the major structural protein of trout CNS myelin 36K was isolated and sequenced. The deduced amino acid sequence did not reveal a putative transmembrane domain and exhibited no structural homology with any of the known myelin proteins. 36K instead shared characteristic structural elements with enzymes of the short-chain dehydrogenase family. The highest similarity in the database (60%), however, was obtained with a human protein of unknown function. By Northern blotting, a single mRNA species of about 2 kb was identified, which was expressed in brain tissue but not in liver. By in situ hybridization, a selective labeling of myelinating glial cells in the trout CNS but not in the PNS was revealed. The developmental appearance of the 36K transcript closely coincided with a period of active myelin deposition in most regions of the trout brain. As a first step in elucidating the structural and biochemical role of 36K for myelin formation and maintenance, we have overexpressed it in Escherichia coli as a soluble His-tag fusion protein and purified it in high yield by Ni+-chelated affinity chromatography. By SDS-PAGE, a single band of the expected molecular size was revealed, which heavily cross-reacted with polyclonal antibodies generated against the native protein. The results of circular dichroism spectroscopy are compatible with a betaalphabeta-barrel structure (Rossman fold), confirming the results of computer-assisted secondary structure predictions.

Growth/differentiation factor-15 (GDF-15), a novel member of the TGF-beta superfamily, promotes survival of lesioned mesencephalic dopaminergic neurons in vitro and in vivo and is induced in neurons following cortical lesioning.

This review summarizes the evidence that GDF-15, a recently discovered member of the TGF-beta superfamily, is a trophic factor for nigral dopamine neurons, both in vitro and in vivo. Specifically, GDF-15 promotes survival and differentiation of embryonic rat dopaminergic neurons, but not of other neuron populations, with the exception of serotonergic raphe neurons. The neurotrophic effect of GDF-15 seems to be direct and not mediated through glial cells. In the rat 6-hydroxydopamine model of parkinsonism GDF-15 rescues intoxicated dopaminergic neurons and abolishes abnormal turning behavior. The most prominent site of synthesis of GDF-15 within the brain is the choroid plexus, which secretes GDF-15 into the cerebrospinal fluid, from where the molecule can penetrate through the ependymal layer into the parenchyma. Analysis of mouse mutants lacking GDF-15 will reveal whether the endogenous factor also has a role in promoting embryonic and protecting lesioned nigral dopamine neurons.

Genetic influence on blood pressure and lipid parameters in a sample of Polish twins.

We studied 76 healthy monozygotic (MZ) and same-sex dizygotic (DZ) twin pairs (mean age 35 +/- 8 years, body mass index, BMI, 23.6 +/- 3.9 kg/m2) to determine genetic and environmental contributions to systolic (SBP) and diastolic (DBP) blood pressure, heart rate (HR) and serum lipids [total cholesterol (TC), low-density lipoprotein cholesterol (LDL-chol), high-density lipoprotein cholesterol (HDL-chol) and triglycerides (TG)I. SBP, DBP and HR were measured clinically and by ambulatory blood pressure monitoring (ABPM). Parameters of the genetic models for age-, sex- and BMI-adjusted data were estimated by model fitting and path analysis technique using LISREL 8. We found significant genetic effect on SBP and DBP for both clinical and ABP measurements, ranging from 37% for night-time ambulatory DBP to 79% for daytime ambulatory SBP. Estimates of genetic effects were higher for daytime than night-time ABP values, and higher for ambulatory 24-h SBP than office SBP measurements, with the reverse true for DBP. Significant genetic effect on HR ranged from 59% for office measurements to 69% for 24-h mean values. In summary, we also found genetic effect on TC, LDL-chol and HDL-chol with estimates ranging from 36% to 64%, but not on TG. Furthermore, a shared environmental component for TG was found, estimated at 36%. We showed significant genetic effect on both office and ambulatory BP and HR, with stronger genetic effect on daytime than night-time BP. We also found genetic effect on TC and lipoprotein fractions, but no significant genetic effect on TG. Environmental factors influencing serum TG, such as alcohol consumption, may explain the apparent lack of genetic effect in this healthy, non-obese population.

Expression of growth differentiation factor-15/ macrophage inhibitory cytokine-1 (GDF-15/MIC-1) in the perinatal, adult, and injured rat brain.

We and others have recently cloned a new member of the transforming growth factor-beta superfamily, growth differentiation factor-15/ macrophage inhibitory cytokine-1 (GDF-15/MIC-1). Using in situ hybridization and immunohistochemistry, we determined the distribution of GDF-15/MIC-1 mRNA and protein in the perinatal and cryolesioned adult rat brain. The choroid plexus epithelium of all ventricles represents the site of strongest and almost exclusive mRNA expression in the normal perinatal and adult brain. The newborn rat brain reveals GDF-15/MIC-1 immunoreactivity (ir) in ependymal cells lining the ventricles, in the striatal subventricular zone, and in populations of nonneural cells of the thalamic/hippocampal lamina affixa, in addition to that in the choroid plexus. Unilateral cryogenic cortical lesioning induced a significant increase of GDF-15/MIC-1 mRNA expression and ir at the lesion site and expression in presumed neurons within the dorsal thalamic area. At the lesion site, GDF-15/MIC-1-producing cells showed immuncytochemical features of neurons, macrophages, and activated microglial cells. Fluorescent microscopy revealed both intra- and extracellular GDF-15/MIC-1 ir. Up-regulation of GDF-15/MIC-1 in activated macrophages (Mstraight phi) is also supported by RT-PCR, ICC, and Western blot experiments showing pronounced induction of GDF-15/MIC-1 expression (mRNA and protein) in retinoic acid/phorbol ester-stimulated human M phi. Our data suggest that 1) GDF-15/MIC-1 is secreted into the cerebrospinal fluid and 2) in the newborn brain may penetrate through the ependymal lining and act on developing neurons and/or glial cells. As a constituent of cells in the lamina affixa, the protein might be involved in the regulation of mesenchyme-epithelial interactions. Finally, GDF-15/MIC-1 may also act within the antiinflammatory cytokine network activated in CNS lesions.

Lack of neurotrophin-4 causes selective structural and chemical deficits in sympathetic ganglia and their preganglionic innervation.

Neurotrophin-4 (NT-4) is perhaps the still most enigmatic member of the neurotrophin family. We show here that NT-4 is expressed in neurons of paravertebral and prevertebral sympathetic ganglia, i.e., the superior cervical (SCG), stellate (SG), and celiac (CG) ganglion. Mice deficient for NT-4 showed a significant reduction (20-30%) of preganglionic sympathetic neurons in the intermediolateral column (IML) of the thoracic spinal cord. In contrast, neuron numbers in the SCG, SG, and CG were unchanged. Numbers of axons in the thoracic sympathetic trunk (TST) connecting the SG with lower paravertebral ganglia were also reduced, whereas axon numbers in the cervical sympathetic trunk (CST) were unaltered. Axon losses in the TST were paralleled by losses of synaptic terminals on SG neurons visualized by electron microscopy. Furthermore, immunoreactivity for the synaptic vesicle antigen SV2 was clearly reduced in the SG and CG. Levels of catecholamines and tyrosine hydroxylase immunoreactivity were dramatically reduced in the SG and the CG but not in the SCG. Despite this severe phenotype in the sympathetic system, blood pressure levels were not reduced and displayed a pattern more typical of deficits in baroreceptor afferents. Numbers of IML neurons were unaltered at postnatal day 4, suggesting a postnatal requirement for their maintenance. In light of these and previous data, we hypothesize that NT-4 provided by postganglionic sympathetic neurons is required for establishing and/or maintaining synapses of IML neurons on postganglionic cells. Impairment of synaptic connectivity may consequently reduce impulse flow, causing a reduction in transmitter synthesis in postganglionic neurons.

Functions of transforming growth factor-beta isoforms in the nervous system. Cues based on localization and experimental in vitro and in vivo evidence.

This review briefly describes the cellular distribution and documented roles of the transforming growth factor (TGF)-beta isoforms TGF-beta2 and -beta3 in the central and peripheral nervous system. TGF-beta2 and -beta3 are coexpressed in developing radial glial and mature astroglial and Schwann cells, as well as in subpopulations of differentiated neurons, most prominently in cortical, hippocampal, and brainstem/spinal cord motor neurons. In vitro studies have suggested a number of potential, physiologically relevant functions for TGF-betas including regulation of astroglial cell proliferation, expression of adhesion molecules, survival promoting roles for neurons in combination with established neurotrophic factors, and differentiative actions on neurons.

Growth/differentiation factor-15/macrophage inhibitory cytokine-1 is a novel trophic factor for midbrain dopaminergic neurons in vivo.

Transforming growth factor-betas (TGF-betas) constitute an expanding family of multifunctional cytokines with prominent roles in development, cell proliferation, differentiation, and repair. We have cloned, expressed, and raised antibodies against a distant member of the TGF-betas, growth/differentiation factor-15 (GDF-15). GDF-15 is identical to macrophage inhibitory cytokine-1 (MIC-1). GDF-15/MIC-1 mRNA and protein are widely distributed in the developing and adult CNS and peripheral nervous systems, including choroid plexus and CSF. GDF-15/MIC-1 is a potent survival promoting and protective factor for cultured and iron-intoxicated dopaminergic (DAergic) neurons cultured from the embryonic rat midbrain floor. The trophic effect of GDF-15/MIC-1 was not accompanied by an increase in cell proliferation and astroglial maturation, suggesting that GDF-15/MIC-1 probably acts directly on neurons. GDF-15/MIC-1 also protects 6-hydroxydopamine (6-OHDA)-lesioned nigrostriatal DAergic neurons in vivo. Unilateral injections of GDF-15/MIC-1 into the medial forebrain bundle just above the substantia nigra (SN) and into the left ventricle (20 microgram each) immediately before a 6-OHDA injection (8 microgram) prevented 6-OHDA-induced rotational behavior and significantly reduced losses of DAergic neurons in the SN. This protection was evident for at least 1 month. Administration of 5 microgram of GDF-15/MIC-1 in the same paradigm also provided significant neuroprotection. GDF-15/MIC-1 also promoted the serotonergic phenotype of cultured raphe neurons but did not support survival of rat motoneurons. Thus, GDF-15/MIC-1 is a novel neurotrophic factor with prominent effects on DAergic and serotonergic neurons. GDF-15/MIC-1 may therefore have a potential for the treatment of Parkinson's disease and disorders of the serotonergic system.

GDF-15/MIC-1 a novel member of the TGF-beta superfamily.

We have cloned, expressed, and raised antibodies against a novel member of the TGF-beta superfamily, growth/differentiation factor-15 (GDF-15). The predicted protein is identical to macrophage inhibitory cytokine-1 (MIC-1), which was discovered simultaneously. GDF-15 is a more distant member of the TGF-beta superfamily and does not belong to one of the known TGF-beta subfamilies. In the CNS, GDF-15/MIC-1 mRNA is abundantly expressed by the choroid plexus. In addition we have preliminary evidence that GDF-15/MIC-1 is a potent trophic factor for selected classes of neurons in vitro and in vivo. Thus, GDF-15 is a novel neurotrophic factor with prospects for the treatment of disorders of the CNS.

GDNF family members and their receptors: expression and functions in two oligodendroglial cell lines representing distinct stages of oligodendroglial development.

Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN), and persephin (PSP) constitute a subfamily of transforming growth factor-betas (TGF-betas) with prominent roles in the regulation of neuron survival and differentiation. Although numerous members of the TGF-beta superfamily are important regulators of glial cell functions in health and disease, it is unknown whether any member of the GDNF subfamily may have functions in normal or pathological glial cell performances. To begin to address this issue, we have studied expression and putative functions of GDNF, NTN, PSP, and their receptors in two cell lines representing models for oligodendrocyte progenitor cells (OLI-neu) and immature oligodendrocytes (OLN-93), respectively. RT-PCR analysis revealed expression of all three growth factor mRNAs in OLI-neu and OLN-93 cells. Expression was weak in OLI-neu cells, while both NTN and PSP mRNAs were strongly expressed in OLN-93 cells. Furthermore, OLI-neu and OLN-93 cells expressed transcripts encoding the GDNF receptors Ret and GFRalpha-1. The two splice variants for GFRalpha-2 were exclusively synthesized in OLI-neu cells. Similarly, primary O-2A progenitor cells and enriched mature oligodendrocytes expressed Ret, GFRalpha-1 and GFRalpha-2 mRNAs. Both GDNF and NTN stimulated DNA synthesis monitored by BrdU incorporation of OLI-neu cells in a dose-dependent fashion. Co-administration of TGF-beta significantly reduced this effect. Similarly, PDGF co-applied with GDNF or NTN down-regulated proliferation in OLI-neu cells. In contrast, OLN-93 cells did not respond to GDNF or NTN with increased incorporation of BrdU. Expression of GDNF, NTN, and their receptors and distinct effects in two model cell lines of oligodendrocyte development suggest that functions of members of the GDNF family and their receptors may not be restricted to neurons and may be implicated in oligodendrocyte development.

GDNF-related factor persephin is widely distributed throughout the nervous system.

Persephin (PSP) is the most recently discovered member of the GDNF family of neurotrophic factors. We have used an RT-PCR approach to start addressing the putative functional significance of PSP by determining sites of its synthesis in the neonatal rat brain. Generally, two transcripts were found. Sequence analysis of the transcripts identifies an 88 bp intronic sequence. Neural tissues analysed included cortex, hippocampus, striatum, diencephalon, mesencephalon, cerebellum, hindbrain and spinal cord as well as superior cervical, dorsal root ganglia, adrenal gland, and PC12 pheochromocytoma cells. As non-neuronal tissues, sciatic nerve, optic nerve, primary astroglial, oligodendroglial, O2A progenitor, and glioma cells (C6, B49) were also included. All tissues/cells except oligodendrocytes and O2A progenitor cells were strongly positive for PSP mRNA. To test the hypothesis of whether PSP might act as a target-derived factor, as suggested for GDNF, the motoneuron-muscle axis has been analysed. PSP is synthesized in skeletal muscle and, to a higher extent, in the spinal cord. Moreover, PSP is synthesized in purified embryonic motoneurons. Together, these data do not support a role for PSP as a typical target-derived neurotrophic factor for motoneurons. We conclude that PSP is synthesized throughout the nervous system and that it is presumably of both astroglial and neuronal origin, in contrast to GDNF and neurturin, which seem to be predominantly of neuronal origin.

The serotonin transporter gene and peer-rated neuroticism.

Polymorphisms in the serotonin transporter gene (5HTT) have been reported to be associated with neuroticism (emotionality) and with depression. A recent report of an association between 5HTT and neuroticism involved unselected samples and self-report questionnaires. We attempted to extend these findings using a selected extremes design and peer ratings. From a sample of 2085 individuals, each assessed on neuroticism by two independent peers, we selected 52 individuals from the top 5% and 54 individuals from the bottom 5%. No association was found for either a functional 44 bp insertion/deletion polymorphism in 5HTT regulatory sequence (5HTTLPR) or for a non-functional variable number tandem repeat 5HTT polymorphism.

Partial characterization of the 5'-flanking region of trout IP: a Po-like gene containing a PLP-like promoter.

The IP gene of trout encodes two Po-like glycoproteins which are expressed by oligodendrocytes in the fish CNS. A 679 bp fragment of its 5'-flanking region was isolated from a genomic library and sequenced. The transcription start point was determined 124 bp upstream the ATG initiator codon by primer extension analysis. Apart from a modified TATA-box and an inverted CCAAT-box located at canonical distances from the transcription start site several eucaryotic cis-acting regulatory elements were identified in the 679 bp upstream region, including an AP-1 binding site, a brain specific Sp1 motif, a cyclic AMP responsive element and a consensus sequence for POU homeodomain protein binding. The occurence of respective DNA-binding proteins for Sp1, AP-1 and POU in the nuclei of trout oligodendrocyte progenitor cells was verified by gel retardation experiments. Functional activity of various subfragments of the 679 bp upstream region was demonstrated by CAT reporter gene analysis. A computer-assisted sequence alignment of the trout IP 5'-flanking end with the corresponding region of the mammalian PLP gene promoter revealed four sites of high homology, while similarity with the mammalian Po gene promotor was low. The results are discussed with respect to the phylogenetic shift from Po-like proteins to PLP during evolution of the vertebrate CNS myelin sheath.