Morphology and transfection study of human microvascular endothelial cell angiogenesis: an in vitro three-dimensional model

Microvascular endothelial cells from human neonatal foreskin were grown in vitro until a three-dimensional network of capillary-like structures was formed. All stages of the angiogenic cascade could be observed in this in vitro model, including the formation of an internal lumen. The microscopy focused on morphology, formation of an internal lumen, role of the extracellular matrix, polarity of the cells, and the time-course of the angiogenic cascade. Bright-field microscopy revealed cells arranged circularly side by side and the internal lumen of capillary-like structures was verified by electron microscopy. Immunolabeling revealed a peritubular localization of collagen IV. Reporter gene expression after the formation of capillary-like structures was marginally higher than control expression, but clearly lower than the expression of cells at the stage of proliferation. Highest transfection efficiencies were obtained using vectors with the CMV promoter and the long fragment of the Ets-1 promoter. This is a first study of transfection efficiencies mapped for stages of in vitro angiogenesis. We describe here the morphological features of a long-term in vitro model of angiogenesis of human microvascular endothelial cells that could be used for transfection studies, without the provision of an extracellular matrix substrate. The cells self-create their own extracellular matrix to proliferate and form a three-dimensional network of capillary-like structures with an internal lumen.     

Caveolin-1 deficiency stimulates neointima formation during vascular injury

Neointima formation is a process characterized by smooth muscle cell (SMC) proliferation and extracellular matrix deposition in the vascular intimal layer. Here, we critically evaluate the role of caveolin-1 (Cav-1) in the pathogenesis of neointima formation. Cav-1 and caveolae organelles are particularly abundant in SMCs, where they are thought to function in membrane trafficking and signal transduction events. To directly evaluate the role of Cav-1 in the pathogenesis of neointimal lesions, we used Cav-1-deficient (Cav-1 -/-) mice as a model system. The right common carotid artery of wild-type and Cav-1 -/- mice was ligated just proximal to its bifurcation. Specimens were then harvested 4-weeks postligation and processed for morphometric and immunohistochemical analyses. The carotids of Cav-1 -/- mice showed significantly more intimal hyperplasia with subtotal luminal obstruction, as compared to wild-type mice. These neointimal lesions consisted mainly of SMCs. Mechanistically, neointimal lesions derived from Cav-1 -/- mice exhibited higher levels of phospho-p42/44 MAP kinase and cyclin D1 immunostaining, consistent with the idea that Cav-1 functions as a negative regulator of signal transduction. A significant increase in phospho-Rb (Ser780) immunostaining was also observed, in line with the upregulation of cyclin D1. In conclusion, using a carotid artery blood-flow cessation model, we show that genetic ablation of Cav-1 in mice stimulates SMC proliferation (neointimal hyperplasia), with concomitant activation of the p42/44 MAP kinase cascade and upregulation of cyclin D1. Importantly, our current study is the first to investigate the role of Cav-1 in SMC proliferation in the vascular system using Cav-1 -/- mice.     

Satellite glial cells in the trigeminal ganglion as a determinant of orofacial neuropathic pain

Satellite glial cells (SGCs) tightly envelop the perikarya of primary sensory neurons in peripheral ganglion and are identified by their morphology and the presence of proteins not found in ganglion neurons. These SGC-unique proteins include the inwardly rectifying K(+) channel Kir4.1, the connexin-43 (Cx43) subunit of gap junctions, the purinergic receptor P2Y4 and soluble guanylate cyclase. We also present evidence that the small-conductance Ca(2+)-activated K(+) channel SK3 is present only in SGCs and that SGCs divide following nerve injury. All the above proteins are involved, either directly or indirectly, in potassium ion (K(+)) buffering and, thus, can influence the level of neuronal excitability, which, in turn, has been associated with neuropathic pain conditions. We used in vivo RNA interference to reduce the expression of Cx43 (present only in SGCs) in the rat trigeminal ganglion and show that this results in the development of spontaneous pain behavior. The pain behavior is present only when Cx43 is reduced and returns to normal when Cx43 concentrations are restored. This finding shows that perturbation of a single SGC-specific protein is sufficient to induce pain responses and demonstrates the importance of PNS glial cell activity in the pathophysiology of neuropathic pain.     

Auxin triggers transient local signaling for cell specification in Arabidopsis embryogenesis

The Arabidopsis embryonic root meristem is initiated by the specification of a single cell, the hypophysis. This event critically requires the antagonistic auxin response regulators MONOPTEROS and BODENLOS, but their mechanism of action is unknown. We show that these proteins interact and transiently act in a small subdomain of the proembryo adjacent to the future hypophysis. Here they promote transport of auxin, which then elicits a second response in the hypophysis itself. Our results suggest that hypophysis specification is not the direct result of a primary auxin response but rather depends on cell-to-cell signaling triggered by auxin in adjacent cells.     

Advanced glycation end products and antioxidant status in nondiabetic and streptozotocin induced diabetic rats: effects of copper treatment

The effects of Cu(II) supplementation on glycemic parameters, advanced glycation end products (AGEs), antioxidant status (glutathione; GSH and total antioxidant capacity; TAOC) and lipid peroxidative damage (thiobarbituric acid-reactive substances, TBARS) were investigated in streptozotocin (STZ) induced diabetic rats. The study was carried out on Wistar albino rats grouped as control (n = 10), CuCl₂ treated (n = 9), STZ (n = 10) and STZ,CuCl₂ treated (n = 9). STZ was administered intraperitoneally at a single dose of 65 mg/kg and CuCl₂, 4 mg copper/kg, subcutaneously, every 2 days for 60 days. At the end of this period, glucose(mg/dl), Cu(μg/dl), TBARS(μmol/l), TAOC(mmol/l) were measured in plasma, GSH(mg/gHb) in erythrocytes and glycated hemoglobin (GHb)(%) in blood. Plasma AGE-peptides(%) were measured by HPLC flow system with spectrofluorimetric and spectrophotometric detectors connected on-line. Data were analyzed by the non-parametric Kruskal–Wallis and Mann–Whitney U test. In the STZ group glucose, GHb and AGE-peptide levels were all significantly higher than the control group (P < 0.01, P < 0.05, and P < 0.01, respectively). CuCl₂ treated group had significantly lower glucose but significantly higher GHb, TAOC and TBARS levels than the control group (P < 0.05, P < 0.001, P < 0.05 and P < 0.001, respectively). STZ,CuCl₂ treated group had significantly higher GHb, TAOC and TBARS levels compared with the control group (P < 0.001, P < 0.05 and P < 0.05, respectively); but only TAOC level was significantly higher than the STZ group (P < 0.01). This experimental study provides evidence that copper intake increases total antioxidant capacity in both nondiabetic and diabetic states. However despite the potentiated antioxidant defence, lipid peroxidation and glycation enhancing effects of CuCl₂ are evident under nondiabetic conditions.     

The genetic architecture of lipoprotein subclasses in Gullah-speaking African American families enriched for type 2 diabetes: The Sea Islands Genetic African American Registry (Project SuGAR)

We sought to partition the genetic and environmental influences on lipoprotein subclasses and identify genomic regions that may harbor genetic variants that influence serum lipoprotein levels in a sample of Gullah-speaking African-Americans. We genotyped 5,974 SNPs in 979 subjects from 418 pedigrees and used the variance component approach to compute heritability estimates, genetic and environmental correlations, and linkage analyses for selected lipoprotein subclasses. The highest heritability estimate was observed for large VLDL particle concentration (0.56 ± 0.14). Mean LDL particle size and small LDL particle concentration (−0.94) had the strongest genetic correlation estimate. The highest logarithm of odds (LOD) score detected (3.0) was on chromosome 6p24 for small LDL particle concentration. The strongest signal, obtained with the reduced sample of diabetic individuals only, was observed on chromosome 20p13 for small LDL particle concentration. The highest bivariate linkage signal (LOD 2.4) was observed on chromosome 6p24 for mean LDL particle size and small LDL particle concentration.jlr Our results suggest a significant genetic contribution to multiple lipoprotein subclasses studied in this sample and that novel loci on chromosomes 6, 10, 16, and 20 may harbor genes contributing to small, atherogenic LDL particle concentration and large, triglyceride-rich VLDL particle concentration.     

Extended bioluminescence resonance energy transfer (eBRET) for monitoring prolonged protein-protein interactions in live cells

Bioluminescence resonance energy transfer (BRET) is an increasingly popular technique for studying protein-protein interactions in live cells. It is particularly suitable for real-time monitoring of such interactions, however, the timescale over which assays can be carried out is currently relatively short (minutes) due to substrate instability. We present a new derivation of the BRET technology, termed 'extended BRET' (eBRET), which now enables protein-protein interactions to be monitored in real-time for many hours. This capability has significant benefits for investigating cellular function over extended timescales, as we have illustrated using the agonist-induced G-protein coupled receptor/beta-arrestin interaction. The potential for studying the modulation of such interactions by agonists, antagonists, inhibitors, dominant negative mutants and co-expressed accessory proteins is substantial. Furthermore, the advantages of eBRET have important implications for the development of high-throughput BRET screening systems, an ever-expanding area of interest for the pharmaceutical industry.     

Extraction of regulatory gene/protein networks from Medline

MOTIVATION: We have previously developed a rule-based approach for extracting information on the regulation of gene expression in yeast. The biomedical literature, however, contains information on several other equally important regulatory mechanisms, in particular phosphorylation, which we now expanded for our rule-based system also to extract. RESULTS: This paper presents new results for extraction of relational information from biomedical text. We have improved our system, STRING-IE, to capture both new types of linguistic constructs as well as new types of biological information [i.e. (de-)phosphorylation]. The precision remains stable with a slight increase in recall. From almost one million PubMed abstracts related to four model organisms, we manage to extract regulatory networks and binary phosphorylations comprising 3,319 relation chunks. The accuracy is 83-90% and 86-95% for gene expression and (de-)phosphorylation relations, respectively. To achieve this, we made use of an organism-specific resource of gene/protein names considerably larger than those used in most other biology related information extraction approaches. These names were included in the lexicon when retraining the part-of-speech (POS) tagger on the GENIA corpus. For the domain in question, an accuracy of 96.4% was attained on POS tags. It should be noted that the rules were developed for yeast and successfully applied to both abstracts and full-text articles related to other organisms with comparable accuracy. AVAILABILITY: The revised GENIA corpus, the POS tagger, the extraction rules and the full sets of extracted relations are available from http://www.bork.embl.de/Docu/STRING-IE