Determinants of circulating calcitriol in cardiovascular disease.

Circulating calcitriol may contribute to the risk of cardiovascular disease (CVD), but its regulation in patients with CVD is poorly characterized. We therefore aimed to assess determinants of circulating calcitriol in these patients. We analyzed 2183 independent samples from a large cohort of patients scheduled for coronary angiography and 1727 independent samples from different other cohorts from patients with a wide range of CVDs, including heart transplant candidates, to quantify the association of different parameters with circulating calcitriol. We performed univariable and multivariable linear regression analyses using the mathematical function that fitted best with circulating calcitriol. In the multivariable analysis of the large single cohort, nine parameters remained significant, explaining 30.0 % (32.4 % after exclusion of 22 potential outliers) of the variation in circulating calcitriol (r=0.548). Log-transformed 25-hydroxyvitamin D [25(OH)D] and log-transformed glomerular filtration rate were the strongest predictors, explaining 17.6 % and 6.6 %, respectively, of the variation in calcitriol. In the analysis of the combined other cohorts, including heart transplant candidates, the multivariable model explained a total of 42.6 % (46.1 % after exclusion of 21 potential outliers) of the variation in calcitriol (r=0.653) with log-transformed fibroblast growth factor-23 and log-transformed 25(OH)D explaining 29.0 % and 6.2 %, respectively. Circulating 25(OH)D was positively and FGF-23 inversely associated with circulating calcitriol. Although significant, PTH was only a weak predictor of calcitriol in both analyses (<2.5 %). In patients with CVD, FGF-23 and 25(OH)D are important independent determinants of circulating calcitriol. The relative importance of these two parameters may vary according to CVD severity. Future studies should focus on the clinical importance of regulating circulating calcitriol by different parameters.

Improvement of fractional flow reserve and collateral flow by treatment with external counterpulsation (Art.Net.-2 Trial).

BACKGROUND: Arteriogenesis (collateral artery growth) is nature's most efficient rescue mechanism to overcome the fatal consequences of arterial occlusion or stenosis. The goal of this trial was to investigate the effect of external counterpulsation (ECP) on coronary collateral artery growth. MATERIALS AND METHODS: A total of 23 patients (age 61 +/- 2.5 years) with stable coronary artery disease and at least one haemodynamic significant stenosis eligible for percutaneous coronary intervention were prospectively recruited into the two study groups in a 2 : 1 manner (ECP : control). One group (ECP group, n = 16) underwent 35 1-h sessions of ECP in 7 weeks. In the control group (n = 7), the natural course of collateral circulation over 7 weeks was evaluated. All patients underwent a cardiac catheterization at baseline and after 7 weeks, with invasive measurements of the pressure-derived collateral flow index (CFIp, primary endpoint) and fractional flow reserve (FFR). RESULTS: In the ECP group, the CFIp (from 0.08 +/- 0.01 to 0.15 +/- 0.02; P < 0.001) and FFR (from 0.68 +/- 0.03 to 0.79 +/- 0.03; P = 0.001) improved significantly, while in the control group no change was observed. Only the ECP group showed a reduction of the Canadian Cardiovascular Society (CCS, P = 0.008) and New York Heart Association (NYHA, P < 0.001) classification. CONCLUSION: In this study, we provide direct functional evidence for the stimulation of coronary arteriogenesis via ECP in patients with stable coronary artery disease. These data might open a novel noninvasive and preventive treatment avenue for patients with non-acute vascular stenotic disease.

Coagulation factor XII (FXII) activity, activated FXII, distribution of FXII C46T gene polymorphism and coronary risk.

BACKGROUND: Whether factor XII (FXII) activity, its 46C>T polymorphism and activated FXII (FXIIa) are associated with coronary heart disease (CHD) remains to be determined. METHODS: FXII, FXIIa and the FXII 46C>T polymorphism were determined in a hospital-based cohort of 2615 patients undergoing coronary angiography. RESULTS: Fifty-seven per cent of the participants were identified as wild-type (46CC), 38% as heterozygous (46CT) and 5% as homozygous (46TT) for FXII 46C>T. FXII and FXIIa levels were significantly lower in carriers of the T-allele: 132 (97-151) U dL(-1) FXII in 46CC, 87 (77-99) U dL(-1) FXII in 46CT and 53 (42-67) U dL(-1) FXII in 46TT carriers (P < 0.001), and 2.8 (2.3-3.5) microg L(-1) FXIIa in CC, 2.1 (1.6-2.6) microg L(-1) FXIIa in CT and 1.2 (0.9-1.5) microg L(-1) FXIIa in TT carriers (P < 0.001; medians, lower and upper quartiles). Patients with stable CHD (n = 935), a history of myocardial infarction (n = 785) or who were suffering from acute coronary syndromes (ACS; n = 323) had significantly lower FXII levels than controls (n = 572). The differences remained statistically significant after adjustments for age, sex, diabetes mellitus, smoking, hypercholesterolemia and hypertension. Significantly reduced FXIIa levels in ACS patients lost significance once adjusted for covariates. FXII genotype was not associated with any clinical phenotype. CONCLUSION: Lower FXII activity represents an independent risk for CHD and ACS. This is not the case for FXIIa levels or the FXII 46C>T variation.

Low grade inflammation in juvenile obesity and type 1 diabetes associated with early signs of atherosclerosis.

BACKGROUND: Subclinical inflammation has been implicated in the initiation and/or progression of atherosclerosis. Diabetes mellitus and obesity are risk factors for atherosclerosis, and asymptomatic low grade inflammation occurs prior to overt vascular lesions in these patients. In contrast to adults, little information exists concerning low grade inflammation in young type 1 diabetes and juvenile obesity. AIM: To investigate low grade inflammation and immune activation in juvenile diabetes mellitus and obesity. METHODS: hs-CRP, soluble interleukin-2 receptor (sIL-2R), C-peptide, insulin, cortisol, vitamin B12, folic acid, leptin, and homocysteine were determined in 148 patients with juvenile type 1 diabetes, 86 obese children and 142 normal weighted age-matched healthy controls. Intima-media thickness (IMT) and lumen diameter of both common carotid arteries (CCA) was measured by ultrasonography in 52 healthy pediatric controls, 10 diabetics, and 34 obese juveniles. RESULTS: Serum hs-CRP was significantly elevated in patients with type 1 diabetes (p < 0.0001), and obese children (p < 0.0001) as compared to the control group. The obese juveniles (p < 0.0001) and the diabetics (p < 0.0001) showed significantly increased values for IMT of CAAs. Levels of homocysteine, sIL-2R, insulin, cortisol, vitamin B12, and folic acid did not differ from the controls. The elevation of hs-CRP was more pronounced in obesity as compared to type 1 diabetes (p < 0.0001), and the hs-CRP values correlated significantly with body mass index standard deviation score (BMI-SDS) values. Furthermore, the IMT and the luminal diameter of CCAs showed significant correlations with BMI-SDS values. CONCLUSION: A low grade inflammation as determined by serum hs-CRP is significantly increased in children with type 1 diabetes, and even more pronounced in apparently healthy juveniles with obesity. The increased IMT of CCAs strongly argues for an association between this low grade inflammation and early atherosclerotic vessel injury.

Differential effects of apolipoprotein E isoforms on phosphorylation at specific sites on tau by glycogen synthase kinase-3 beta identified by nano-electrospray mass spectrometry.

Previously published data have shown an allele-specific variation in the in vitro binding of apolipoprotein E (apoE) to tau, which prompted the hypothesis that apoE binding may protect tau from phosphorylation, apoE3 being more efficient than apoE4. We have, therefore, investigated the effects of apoE on tau phosphorylation in vitro by the proline-directed kinase, glycogen synthase kinase (GSK)-3 beta. The phosphopeptide maps of tau alone, of tau with apoE3 and of tau with apoE4 were very similar. When apoE2 was present a further four spots were evident. Additionally, of the 15 peptides phosphorylated in the presence or absence of apoE, subtle differences, some isoform-specific, in the relative amounts of phosphorylation were observed.

FGF4 dissociates anti-tumorigenic from differentiation signals of retinoic acid in human embryonal carcinomas.

A subset of male germ cell cancers presenting with advanced stage abundantly express the fibroblast growth factor-4 (FGF4). FGF4 expression is restricted in vitro to undifferentiated embryonal carcinomas (ECs). During induced differentiation, FGF4 expression is repressed in maturation sensitive but not resistant human ECs, suggesting FGF4 plays an important role in malignant growth or differentiation of ECs. To explore these FGF4 signals in male germ cell cancers, the multipotent human EC NTERA-2 clone D1 (NT2/D1) cell line was studied. All-trans-retinoic acid (RA)-treatment of these cells induces a neuronal phenotype and represses tumorigenicity and FGF4 expression. In contrast, RA-treatment of retinoid resistant lines derived from NT2/D1 cells failed to repress FGF4 expression. This implicated FGF4 directly in regulating human EC growth or differentiation. To evaluate further this FGF4 role, FGF4 was constitutively over-expressed in NT2/D1 cells using a CMV-driven expression vector containing the neomycin resistance gene. Three stable transfectants expressing exogenous FGF4 were studied as was a control transfectant only expressing the neomycin resistance gene. RA-treatment repressed endogenous but not exogenous FGF4 expression. RA-treatment of these transfectants induced morphologic and immunophenotypic maturation, changes in RA-regulated genes, and a G1 cell cycle arrest in a manner similar to parental NT2/D1 cells. This indicated FGF4 over-expression did not block RA-mediated differentiation. As expected, RA-treatment repressed tumorigenicity of the control transfectant after subcutaneous injection into athymic mice. Despite RA-treatment, this repressed tumorigenicity was overcome in all the transfectants over-expressing FGF4. The histopathology and neovascularization did not appreciably differ between xenograft tumors derived from FGF4 over-expressing versus control transfectants. FGF4 expression studies were extended to patient-derived germ cell tumors using total cellular RNA Northern analysis and an immunohistochemical assay developed to detect FGF4 protein expression. Germ cell tumors with EC components were significantly more likely to express FGF4 mRNA (P < or = 0.0179) than other examined germ cell tumors without EC components. Immunohistochemical results from 43 germ cell tumors demonstrated increased FGF4 expression especially in non-seminomas having EC components. Thus, FGF4 promotes directly malignant growth of cultured ECs, overcomes the antitumorigenic actions of RA, and is selectively expressed in specific histopathologic subsets of germ cell tumors. Taken together, these findings indicate how differentiation and anti-tumorigenic retinoic acid signals can be dissociated in germ cell cancer.

Molecular mechanisms that underlie structural and functional changes at the postsynaptic membrane during synaptic plasticity.

The synaptic plasticity that is addressed in this review follows neurodegeneration in the brain and thus has both structural as well as functional components. The model of neurodegeneration that has been selected is the kainic acid lesioned hippocampus. Degeneration of the CA3 pyramidal cells results in a loss of the Schaffer collateral afferents innervating the CA1 pyramidal cells. This is followed by a period of structural plasticity where new synapses are formed. These are associated with changes in the numbers and shapes of spines as well as changes in the morphometry of the dendrites. It is suggested that this synaptogenesis is responsible for an increase in the ratio of NMDA to AMPA receptors mediating excitatory synaptic transmission at these synapses. Changes in the temporal and spatial properties of these synapses resulted in an altered balance between LTP and LTD. These properties together with a reduction in the inhibitory drive increased the excitability of the surviving CA1 pyramidal cells which in turn triggered epileptiform bursting activity. In this review we discuss the insights that may be gained from studies of the underlying molecular machinery. Developments in one of the collections of the cogs in this machinery has been summarized through recent studies characterizing the roles of neural recognition molecules in synaptic plasticity in the adult nervous systems of vertebrates and invertebrates. Such investigations of neural cell adhesion molecules, cadherins and amyloid precursor protein have shown the involvement of these molecules on the morphogenetic level of synaptic changes, on the one hand, and signal transduction effects, on the other. Further complex cogs are found in the forms of the low-density lipoprotein receptor (LDL-R) family of genes and their ligands play pivotal roles in the brain development and in regulating the growth and remodelling of neurones. Evidence is discussed for their role in the maintenance of cognitive function as well as Alzheimer's. The molecular mechanisms responsible for the clustering and maintenance of transmitter receptors at postsynaptic sites are the final cogs in the machinery that we have reviewed. Postsynaptic densities (PSD) from excitatory synapses have yielded many cytoskeletal proteins including actin, spectrin, tubulin, microtubule-associated proteins and calcium/calmodulin-dependent protein kinase II. Isolated PSDs have also been shown to be enriched in AMPA, kainate and NMDA receptors. However, recently, a new family of proteins, the MAGUKs (for membrane-associated guanylate kinase) has emerged. The role of these proteins in clustering different NMDA receptor subunits is discussed. The MAGUK proteins are also thought to play a role in synaptic plasticity mediated by nitric oxide (NO). Both NMDA and non-NMDA receptors are highly clustered at excitatory postsynaptic sites in cortical and hippocampal neurones but have revealed differences in their choice of molecular components. Both GABAA and glycine (Gly) receptors mediate synaptic inhibition in the brain and spinal cord. Whilst little is known about how GABAA receptors are localized in the postsynaptic membrane, considerable progress has been made towards the elucidation of the molecular mechanisms underlying the formation of Gly receptors. It has been shown that the peripheral membrane protein gephyrin plays a pivotal role in the formation of Gly receptor clusters most likely by anchoring the receptor to the subsynaptic cytoskeleton. Evidence for the distribution as well as function of gephyrin and Gly receptors is discussed. Postsynaptic membrane specializations are complex molecular machinery subserving a multitude of functions in the proper communication between neurones. Despite the fact that only a few key players have been identified it will be a fascinating to watch the story as to how they contribute to structural and functional plasticity unfold.

Common defects of different retinoic acid resistant promyelocytic leukemia cells are persistent telomerase activity and nuclear body disorganization.

The acute promyelocytic leukemia (APL) t(15;17) rearrangement fuses the promyelocytic leukemia (PML) gene to the retinoic acid receptor-alpha (RAR alpha). There is expression of the chimeric transcript, PML/RAR alpha, in these APL cells. These clinical APL cases respond to the differentiation agent all-trans retinoic acid (ATRA) with complete but not durable remissions because ATRA resistance develops. The NB4 APL cell line expresses PML/RAR alpha and responds to the growth inhibitory and differentiation-inducing signals of ATRA. To identify mechanisms responsible for ATRA resistance in APL, ATRA-resistant NB4 cell lines were derived from parental NB4 cells using different strategies. These lines were resistant to the growth inhibition and differentiation effects of ATRA. ATRA-resistant cells were isolated as a de novo resistant line from parental NB4 cells (NB4-R1), following chemical mutagenization and selection in ATRA (NB4-R2), or after chronic selection in ATRA (NB4-R3). Common defects linked to this ATRA resistance were found. When cultured in ATRA, these resistant cells still express PML, RAR alpha, and PML/RAR alpha proteins. Sequence abnormalities were not detected in the RAR alpha DNA binding domains cloned from a representative RA-resistant NB4 line. In ATRA-sensitive but not ATRA-resistant NB4 cells, ATRA down-regulated retinoid X receptor-alpha (RXR alpha) expression, a known marker of ATRA response in parental NB4 cells. Notably, engineered overexpression of RXR alpha in ATRA-sensitive NB4 cells did not block ATRA-mediated growth suppression. ATRA treatment of these resistant NB4 lines did not signal a decline in telomerase activity or reorganization of PML-associated nuclear bodies, but both events occurred in ATRA-sensitive NB4 cells. These ATRA-resistant NB4 lines are not fully differentiation-defective, since monocytic maturation was induced following treatment with phorbol 12-myristate 13-acetate (PMA) and 1,25 dihydroxy vitamin D3 (vitamin D3). Notably, induced monocytic differentiation of these distinct ATRA-resistant APL lines markedly repressed telomerase activity. Thus, this study suggests that persistent telomerase activity and nuclear body disorganization are linked to ATRA resistance in APL.

Derivation and characterization of retinoid-resistant human embryonal carcinoma cells.

The retinoids exert potent growth and differentiation effects on normal and neoplastic cells through two families of nuclear receptors. These are the retinoic acid receptors (RAR alpha, RAR beta, RAR gamma) and the retinoid-X receptors (RXR alpha, RXR beta, RXR gamma). All-trans retinoic acid (RA) induces terminal neuronal differentiation and represses tumorigenicity of the multipotent human embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1). Hexamethylene bisacetamide (HMBA) induces a phenotype distinct from RA-treated NT2/D1 cells. This study reports the derivation and characterization of RA- and HMBA-resistant NT2/D1 clones. Nine RA-resistant (NT2/D1-R1 through NT2/D1-R9) and one HMBA-resistant (NT2/D1-H1) clones were derived after mutagen treatment of NT2/D1 cells and selection in RA or HMBA. NT2/D1-R cells were cross-resistant to 9-cis retinoic acid (9-cis RA), a ligand activating the RAR and RXR pathways, but retained maturation response to HMBA. A representative RA-resistant clone, NT2/D1-R1, overcame the antitumorigenic actions of RA as assessed in athymic mice. NT2/D1-H1 cells were dually resistant to RA and 9-cis RA. All these retinoid resistant cells exhibit deregulated expression of RAR gamma but not RAR alpha or RAR beta. Southern analysis using RAR gamma probes shows no apparent structural differences in genomic DNA between NT2/D1 cells and the RA-resistant subclones. Pulsed-field gel electrophoresis (PFGE) with RAR gamma probes demonstrated an Mlu-I restriction fragment length polymorphism, but no other structural abnormalities in these cells or a panel of germ cell tumor (GCT) cell lines. Full-length RAR gamma 1 coding region cDNAs were cloned from NT2/D1 and NT2/D1-R1 cells and these sequences were identical, suggesting RA resistance in these cells is due to altered regulation of RAR gamma. These differentiation-resistant cells are useful to study RAR gamma target genes or mechanisms engaged by these differentiation inducing agents in human embryonal carcinomas.

All-trans-retinoic acid and hexamethylene bisacetamide (HMBA) regulate TGF-alpha and Hst-1/kFGF expression in differentiation sensitive but not in resistant human teratocarcinomas.

The multipotent human teratocarcinoma (TC) cell NTera-2 clone D1 (abbreviated NT2/D1) differentiates into a neuronal lineage after retinoic acid (RA) treatment and a distinct phenotype after hexamethylene bisacetamide (HMBA) treatment. We previously reported that RA treatment of NT2/D1 cells reduces cellular cloning efficiency and nude mouse tumorigenicity. This accompanied a loss of mRNA expression of transforming growth factor-alpha (TGF-alpha) and the fibroblast growth factor kFGF, also known as hst-1 (abbreviated hst-1/kFGF). This study extends prior work by reporting that the distinct phenotype induced by HMBA also decreases cloning efficiency, tumorigenicity, and TGF-alpha and hst-1/kFGF mRNA expression in NT2/D1 cells. These RNA findings were confirmed by measurements of growth factor protein in the conditioned media of inducer-treated and untreated NT2/D1 cells. In two established human TC lines refractory to the actions of RA, N2102ep and Tera-1, RA fails to decrease expression of either growth factor despite induction of its nuclear receptor, RAR-beta. However, HMBA induces morphologic maturation and down-regulation of these growth factors in N2102ep cells. This indicates that the loss of TGF-alpha and hst-1/kFGF expression serves as a new marker of differentiation in human TCs. To explore the effects of these growth factors on growth and differentiation of NT2/D1 cells, TGF-alpha or hst-1/kFGF protein was added following inducer treatment or no treatment. Neither growth factor blocked immunophenotypic differentiation, but both promoted the growth of uninduced NT2/D1 cells in cloning assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Over-expression of transforming growth factor alpha antagonizes the anti-tumorigenic but not the differentiation actions of retinoic acid in a human teratocarcinoma cell.

All-trans retinoic acid (RA) treatment of the multipotent human teratocarcinoma (TC) cell line NTERA-2 clone D1 (abbreviated NT2/D1) induces a neuronal phenotype and other cell lineages. NT2/D1 cells basally express transforming growth factor alpha (TGF-alpha) mRNA and secreted protein. After RA-treatment TGF-alpha expression is markedly reduced. This decline in TGF-alpha expression accompanies the induction of the neuronal phenotype and a marked reduction of tumorigenicity in athymic mice. This suggested a causal link between reduced TGF-alpha expression and the induced differentiation or loss of tumorigenicity of these RA-treated TC cells. To evaluate this possibility, an RA-refractory NT2/D1 subclone was analysed. This subclone, designated NT2/D1-R1, failed to induce differentiation or to decrease TGF-alpha expression despite RA treatment. To further explore the relationship between TGF-alpha expression and RA actions in this human TC cell, a TGF-alpha cDNA was stably transfected and expressed in NT2/D1 cells. RA-treatment of independently obtained TGF-alpha over-expressing clones and a representative control transfectant only expressing the neomycin resistance gene produced a neuronal phenotype similar to parental NT2/D1 cells as assessed by morphologic, immunophenotypic, and gene expression markers of differentiation. RA-treatment of these clones also induced a G1 arrest similar to parental cells. However, only the TGF-alpha over-expressing clones that secreted high levels of TGF-alpha protein into the conditioned media before and after RA treatment still developed tumors in athymic mice despite prior exposure to these cells to RA. This finding demonstrates that TGF-alpha can inhibit the anti-tumorigenic effects of RA in human TCs. Thus, over-expression of a single growth factor that normally declines with RA treatment antagonizes the anti-tumorigenic but not the differentiation actions of RA in this human tumor cell.

Rapid and simple subtyping of the HLA-DRB3 gene in Graves' disease by using temperature-gradient gel electrophoresis.

A HLA-DRB3-subtyping system that uses TGGE for analyzing DRB3 alleles was developed. The polymorphic second exon of the HLA-DRB3 gene was amplified from four homozygous typing cell lines, 223 healthy individuals, and 102 patients with Graves' disease by using the PCR. The PCR products were electrophoresed in a temperature gradient from 35 degrees C to 70 degrees C, and the resulting fragments were visualized by silver staining. Four DRB3 alleles (HLA-DRB3*0101, *0201, *0202, and *0301) were distinguished from one another by the migration of the corresponding homoduplex with the exception that DRB3*0201 was indistinguishable from DRB3*0202. The latter two alleles, however, were resolved by the artificial heteroduplexing approach. Arginine in position 74 of the DRB3 gene product (i.e., HLA-DRB3*0101) was significantly more frequent in Graves' patients than in controls. The relative risk conferred by the presence of the DRB3*0101 was 15.8 (p < or = 0.001). The presence of arginine in position 74 contributed to an etiologic fraction of 75% in our study population. The PCR-TGGE technique is a simple, nonisotopic method, which may be useful in rapid screening of large populations for HLA disease markers.

Amplification and direct sequencing of a cDNA encoding human cytosolic 3-hydroxy-3-methylglutaryl-coenzyme A synthase.

Cytosolic 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) synthase (E.C. 4.1.3.5) is a highly regulated enzyme involved in isoprenoid biosynthesis and therefore a potential target for cholesterol-lowering drugs. Up to now, primary structure data have only been available for chicken, rat and hamster HMG-CoA synthase. Using in vitro amplification and direct sequencing, we have determined the nucleotide sequence of the coding region of the human cytosolic 3-hydroxy-3-methylglutaryl CoA synthase cDNA.