The role of the GABAergic system on insomnia.

Sleep is an essential activity for the survival of mammals. Good sleep quality helps promote the performance of daily functions. In contrast, insufficient sleep reduces the efficiency of daily activities, causes various chronic diseases like Alzheimer's disease, and increases the risk of having accidents. The GABAergic system is the primary inhibitory neurotransmitter system in the central nervous system. It transits the gamma-aminobutyric acid (GABA) neurotransmitter via GABAA and GABAB receptors to counterbalance excitatory neurotransmitters, such as glutamate, noradrenaline, serotonin, acetylcholine, orexin, and dopamine, which release and increase arousal activities during sleep. Several studies emphasized that dysfunction of the GABAergic system is related to insomnia, the most prevalent sleep-related disorder. The GABAergic system comprises the GABA neurotransmitter, GABA receptors, GABA synthesis, and degradation. Many studies have demonstrated that GABA levels correlate with sleep quality, suggesting that modulating the GABAergic system may be a promising therapeutic approach for insomnia. In this article, we highlight the significance of sleep, the classification and pathology of insomnia, and the impact of the GABAergic system changes on sleep. In addition, we also review the medications that target the GABAergic systems for insomnia, including benzodiazepines (BZDs), non-BZDs, barbiturates, GABA supplements, and Chinese herbal medicines.

A three-kilobase fragment of the human Robo4 promoter directs cell type-specific expression in endothelium.

Robo4, a member of the roundabout family, is expressed exclusively in endothelial cells and has been implicated in endothelial cell migration and angiogenesis. Here we report the cloning and characterization of the human Robo4 promoter. The 3-kb 5'-flanking region directs endothelial cell-specific expression in vitro. Deletion and mutation analyses revealed the functional importance of two 12-bp palindromic DNA sequences at -2528 and -2941, 2 SP1 consensus motifs at -42 and -153, and an ETS consensus motif at -119. In electrophoretic mobility shift assays using supershifting antibodies, the SP1 motifs bound SP1 protein, whereas the ETS site bound a heterodimeric member of the ETS family, GA binding protein (GABP). These DNA-protein interactions were confirmed by chromatin immunoprecipitation assays. Transfection of primary human endothelial cells with small interfering RNA against GABP and SP1 resulted in a significant (approximately 50%) reduction in endogenous Robo4 mRNA expression. The 3-kb Robo4 promoter was coupled to LacZ, and the resulting cassette was introduced into the Hprt locus of mice by homologous recombination. Reporter gene activity was observed in the vasculature of adult organs (particularly in microvessels), tumor xenografts, and embryos, where it colocalized with the endothelial cell-specific marker CD31. LacZ mRNA levels in adult tissues and tumors correlated with mRNA levels for endogenous Robo4, CD31, and vascular endothelial cadherin. Moreover, the pattern of reporter gene expression was similar to that observed in mice in which LacZ was knocked into the endogenous Robo4 locus. Together, these data suggest that 3-kb upstream promoter of human Robo4 contains information for cell type-specific expression in the intact endothelium.

A novel class of vascular endothelial growth factor-responsive genes that require forkhead activity for expression.

Recently, we have shown that transient phosphorylation and inhibition of the pro-apoptotic transcription factor, forkhead, by vascular endothelial growth factor (VEGF) is essential for endothelial cell (EC) survival and proliferation. The goal of the present study was to determine whether forkhead (FKHR) also plays a positive role in agonist-mediated gene induction. Human coronary artery ECs were transduced with adenovirus overexpressing constitutively active phosphorylation-resistant triple mutant FKHR or transfected with small interference RNA (siRNA) against FKHR. The cells were then treated in the absence or presence of VEGF and assayed for gene expression using quantitative real-time PCR and Northern blots analyses. The data revealed a novel set of VEGF-responsive genes that require FKHR activity for optimal expression in ECs, including bone morphogenic protein 2, cbp/p300-interacting transactivator 2, decay accelerating factor (DAF), vascular cell adhesion molecule-1 (VCAM-1), manganese superoxide dismutase, endothelial-specific molecule-1, RING1 and YY1-binding protein, and matrix metalloproteinase-10. Consistent with a positive role for FKHR in mediating VEGF induction of DAF and VCAM-1 mRNA, siRNA against FKHR attenuated the effect of VEGF on complement-mediated EC lysis and monocyte adhesion, respectively. VEGF induction of the forkhead-dependent genes was down-regulated by the NF-kappaB inhibitor, constitutively active Ad-IkappaB, and in some cases by the nuclear factor of activated T-cells (NF-AT) inhibitor, cyclosporin. Together, these findings suggest that the VEGF-forkhead signaling axis plays an important functional role in ECs beyond the regulation of cell survival/apoptosis and cell cycle.

Vascular endocan is preferentially expressed in tumor endothelium.

Endothelial cell phenotypes are differentially regulated between different sites of the vascular tree. We tested the hypothesis that endocan, a novel soluble dermatan sulfate proteoglycan, is differentially expressed in the intact endothelium and that site-specific expression is mediated by signals in the local microenvironment. Using a combination of Northern blot analyses, Taqman RT-PCR, and in situ hybridizations, endocan was shown to be preferentially expressed in the endothelial lining of tumor xenografts, including human non-small cell lung cancer, rat glioma, and human renal cell carcinoma. In contrast, endocan mRNA was expressed at low levels in embryos between E4.5 and E18.5. Under in vitro conditions, endocan expression in human umbilical vein endothelial cells (HUVEC) was upregulated by tumor cell-conditioned medium, an effect that was inhibited by the addition of neutralizing antibody to vascular endothelial growth factor (VEGF). Moreover, treatment of HUVEC with VEGF resulted in a dose- and time-dependent increase in endocan mRNA. The results suggest that endocan is preferentially expressed in tumor endothelium in vivo and that its expression is regulated by tumor-derived factors.

Vascular endothelial growth factor is an important determinant of sepsis morbidity and mortality.

Sepsis, the systemic inflammatory response to infection, is a leading cause of morbidity and mortality. The mechanisms of sepsis pathophysiology remain obscure but are likely to involve a complex interplay between mediators of the inflammatory and coagulation pathways. An improved understanding of these mechanisms should provide an important foundation for developing novel therapies. In this study, we show that sepsis is associated with a time-dependent increase in circulating levels of vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) in animal and human models of sepsis. Adenovirus-mediated overexpression of soluble Flt-1 (sFlt-1) in a mouse model of endotoxemia attenuated the rise in VEGF and PlGF levels and blocked the effect of endotoxemia on cardiac function, vascular permeability, and mortality. Similarly, in a cecal ligation puncture (CLP) model, adenovirus-sFlt-1 protected against cardiac dysfunction and mortality. When administered in a therapeutic regimen beginning 1 h after the onset of endotoxemia or CLP, sFlt peptide resulted in marked improvement in cardiac physiology and survival. Systemic administration of antibodies against the transmembrane receptor Flk-1 but not Flt-1 protected against sepsis mortality. Adenovirus-mediated overexpression of VEGF but not PlGF exacerbated the lipopolysaccharide-mediated toxic effects. Together, these data support a pathophysiological role for VEGF in mediating the sepsis phenotype.

Permeability properties of tumor surrogate blood vessels induced by VEGF-A.

Malignant tumors generate new blood vessels by secreting growth factors, particularly members of the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) family. Overall, the new blood vessels that form are hyperpermeable to plasma proteins, a property that is thought to be important for generating new stroma. However, tumor blood vessels are structurally heterogeneous and include microvessels of at least the following distinct types: mother vessels (MV), glomeruloid microvascular proliferations (GMP), arterio-venous-like vascular malformations and capillaries. Our goal was to determine whether macromolecular tracers leaked from all or from only a subset of these vessel types and to elucidate the extravasation pathways. As blood vessels are only a minor component of tumors, and therefore, difficult to study in situ, we used an adenoviral vector to express VEGF-A164, the most important member of the VPF/VEGF family, in mouse tissues. So expressed, VEGF-A164 induces large numbers of surrogate vessels of each type found in tumors in a highly reproducible manner. Overall permeability to plasma proteins was assessed qualitatively with Evan's blue dye and quantitatively with a dual tracer method employing radioactive albumin. Leaky vessels were identified by confocal microscopy (FITC-dextran) and by electron microscopy (ferritin). MV, and to a lesser extent GMP, were found to be hyperpermeable but capillaries and vascular malformations were not. Ferritin extravasated primarily by two trans-cellular routes, vesiculo-vacuolar organelles (VVOs) and fenestrae. This occurred despite a considerable reduction in VVO frequency as VVO membranes translocated to the plasma membrane during MV formation. However, reduction in the number and complexity of VVOs was offset by extensive endothelial cell thinning and a greatly shortened extravasation pathway. Extrapolating these findings to tumors predicts that only a subset of tumor vessels, MV and GMP, is hyperpermeable, and that measures of overall vessel permeability greatly underestimate the permeability of individual MV and GMP.

Preconditioning of primary human endothelial cells with inflammatory mediators alters the "set point" of the cell.

Endothelial cells are highly sensitive to changes in the extracellular milieu. Sepsis results in activation of inflammatory and coagulation pathways. We hypothesized that sepsis-associated mediators may alter the response capacity (so-called "set point") of endothelial cells. Human umbilical vein endothelial cells (HUVEC) were preincubated in the presence or absence of tumor necrosis factor (TNF)-alpha, lipopolysaccharide (LPS), hypoxia, hyperthermia, and/or high glucose; treated with or without thrombin for 4 h; and then processed for RNase protection assays of selected activation markers. Priming with TNF-alpha and LPS significantly inhibited thrombin-mediated induction of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, tissue factor, and E-selectin, but not platelet-derived growth factor-A or CD44. In electrophoretic mobility shift assays, thrombin-treated HUVEC demonstrated inducible binding of p65 NF-kappaB, an effect that was significantly blunted by pretreatment of cells with TNF-alpha and LPS. Consistent with these results, TNF-alpha and LPS attenuated the effect of thrombin on IkappaB phosphorylation, total cytoplasmic IkappaB, and nuclear translocation of p65 NF-kappaB. The inhibitory effect of TNF-alpha on thrombin signaling persisted for up to 24 h following removal of the cytokine. Taken together, these data suggest that inflammatory mediators prime endothelial cells to modulate subsequent thrombin response.

Quantitative multi-gene transcriptional profiling using real-time PCR with a master template.

We previously reported a method for quantitative multi-gene transcriptional profiling with gene-specific standard curves using real-time PCR. Here, we report an approach that increases experimental throughput by using a master template to generate a single standard curve for the estimation of mRNA copy numbers from all genes. We prepared fifty-nine different templates and measured eNOS mRNA copy numbers in Matrigel VEGF-transfectant samples. The copy numbers obtained using each of the fifty-nine templates were within 50% of the copy number obtained using the previously reported method. Analysis of primer design parameters, and subsequent tests, showed that eliminating complementarities between the first nucleotides at the 5'-ends of the forward and reverse primers reduces the influence of saturation effects and produces copy numbers similar to those generated with gene-specific templates-generally, within 20%. Measurements on a panel consisting of eNOS, iNOS, and nNOS further validated the master-template approach. The master-template approach enables rapid quantification of mRNA abundances from panels of hundreds of genes, and will be a valuable tool for screening large numbers of genes as part of a search for biomarkers, the validation of DNA-microarray data, or research into the dynamics of the gene-protein network.

Mechanical stretch is a highly selective regulator of gene expression in human bladder smooth muscle cells.

Application of mechanical stimuli has been shown to alter gene expression in bladder smooth muscle cells (SMC). To date, only a limited number of "stretch-responsive" genes in this cell type have been reported. We employed oligonucleotide arrays to identify stretch-sensitive genes in primary culture human bladder SMC subjected to repetitive mechanical stimulation for 4 h. Differential gene expression between stretched and nonstretched cells was assessed using Significance Analysis of Microarrays (SAM). Expression of 20 out of 11,731 expressed genes ( approximately 0.17%) was altered >2-fold following stretch, with 19 genes induced and one gene (FGF-9) repressed. Using real-time RT-PCR, we tested independently the responsiveness of 15 genes to stretch and to platelet-derived growth factor-BB (PDGF-BB), another hypertrophic stimulus for bladder SMC. In response to both stimuli, expression of 13 genes increased, 1 gene (FGF-9) decreased, and 1 gene was unchanged. Six transcripts (HB-EGF, BMP-2, COX-2, LIF, PAR-2, and FGF-9) were evaluated using an ex vivo rat model of bladder distension. HB-EGF, BMP-2, COX-2, LIF, and PAR-2 increased with bladder stretch ex vivo, whereas FGF-9 decreased, consistent with expression changes observed in vitro. In silico analysis of microarray data using the FIRED algorithm identified c-jun, AP-1, ATF-2, and neurofibromin-1 (NF-1) as potential transcriptional mediators of stretch signals. Furthermore, the promoters of 9 of 13 stretch-responsive genes contained AP-1 binding sites. These observations identify stretch as a highly selective regulator of gene expression in bladder SMC. Moreover, they suggest that mechanical and growth factor signals converge on common transcriptional regulators that include members of the AP-1 family.

N-acetyl-cysteine promotes angiostatin production and vascular collapse in an orthotopic model of breast cancer.

The antioxidant N-acetyl-cysteine (NAC) has been shown to be chemopreventive in clinical studies, and in recent studies, has shown promise in preventing tumor progression. Although the effects of NAC on tumorigenesis have been associated with decreased angiogenesis, the mechanism of the anti-angiogenic activity has not been determined. In the following study, we describe a novel mechanism whereby NAC therapy blocks MDA-MB-435 breast carcinoma cell proliferation and metastasis in an in vivo tumorigenic model. Athymic nude mice bearing MDA-MB-435 xenografts were treated with systemic NAC daily for 8 weeks. NAC treatment resulted in endothelial cell apoptosis and reduction of microvascular density within the core of the tumor leading to significant tumor cell apoptosis/necrosis. Angiostatin accumulated in tumors from NAC-treated but not control animals. Additional studies using a vascular endothelial growth factor-dependent chicken chorioallantoic membrane angiogenic assay recapitulated NAC-induced endothelial apoptosis and coordinate production of angiostatin, a potent endothelial apoptotic factor. In vitro studies showed angiostatin was formed in endothelial cultures in a vascular endothelial growth factor- and NAC-dependent manner, a process that requires endothelial cell surface plasminogen activation. These results suggest that systemic NAC therapy promotes anti-angiogenesis through angiostatin production, resulting in endothelial apoptosis and vascular collapse in the tumor.

Transforming growth factor beta1 induction of vascular endothelial growth factor receptor 1: mechanism of pericyte-induced vascular survival in vivo.

Degeneration of vessels precedes and precipitates the devastating ischemia of many diseases, including retinopathy of prematurity and diabetic retinopathy. Ischemia then leads to proliferative retinopathy and blindness. Understanding the mechanisms of blood vessel degeneration is critical to prevention of these diseases. Vessel loss is associated with oxygen-induced suppression of vascular endothelial growth factor (VEGF) and with pericyte (vascular smooth muscle cell) dropout. The molecular mechanism of pericyte protection of the vasculature is unknown. We show that transforming growth factor beta1 (TGF-beta1)-expressing pericytes are specifically found on vessels resistant to oxygen-induced loss. TGF-beta1 potently induces VEGF receptor 1 (VEGFR-1) expression in endothelial cells and thereby prevents oxygen-induced vessel loss in vivo. Vessel survival is further stimulated with a VEGFR-1-specific ligand, placental growth factor 1. TGF-beta1 induction of VEGFR-1 in endothelial cells explains pericyte protection of vessels and the selective vulnerability of neonatal vessels to oxygen. These results implicate induction and activation of VEGFR-1 as critical targets to prevent vessel loss.

Adipose tissue growth and regression are regulated by angiopoietin-1.

Adipose tissue is unique in its plasticity, capacity for vascular remodeling, and susceptibility to angiogenesis inhibitors. We hypothesize that these characteristics are enabled by maintaining relatively immature adipose vessels to facilitate vascular/tissue remodeling. We examined the vascular maturation regulators, angiopoietin-1, angiopoietin-2, and tie2 receptor, under different weight-modifying conditions. Adipocytes expressed angiopoietin-1, while adipose endothelial cells expressed angiopoietin-2 and tie2. Adipose tissue growth/regression were associated with decreased angiopoietin-1 mRNA and protein, and tie2 phosphorylation. Angiopoietin-2 and tie2 mRNA levels were stable. Angiopoietin-1 mRNA levels inversely correlated with the rates of change in body weight, independent of the direction (weight gain, loss) or etiology (TNP-470, leptin, and diet restriction) of the weight shift. Obese mice injected with ang1/pcDNA had reduced rates of weight gain and fat pad weights, regardless of the route of plasmid administration (subcutaneous, intramuscular, and intravenous). Thus, angiopoietin-1 may regulate adipose tissue growth, suggesting that vascular maturation alters tissue plasticity.

Selective stimulation of VEGFR-1 prevents oxygen-induced retinal vascular degeneration in retinopathy of prematurity.

Oxygen administration to immature neonates suppresses VEGF-A expression in the retina, resulting in the catastrophic vessel loss that initiates retinopathy of prematurity. To investigate the mechanisms responsible for survival of blood vessels in the developing retina, we characterized two VEGF-A receptors, VEGF receptor-1 (VEGFR-1, also known as Flt-1) and VEGF receptor-2 (VEGFR-2, also known as Flk-1). Surprisingly, these two VEGF-A receptors differed markedly during normal retinal development in mice. At 5 days postpartum (P5), VEGFR-1 protein was colocalized with retinal vessels, whereas VEGFR-2 was detected only in the neural retina. Real-time RT-PCR identified a 60-fold induction of VEGFR-1 mRNA in retina from P3 (early vascularization) to P26 (fully vascularized), and no significant change in VEGFR-2 mRNA expression. Placental growth factor-1 (PlGF-1), which exclusively binds VEGFR-1, decreased hyperoxia-induced retinal vaso-obliteration from 22.2% to 5.1%, whereas VEGF-E, which exclusively binds VEGFR-2, had no effect on blood vessel survival. Importantly, under the same conditions, PlGF-1 did not increase vasoproliferation during (a). normal vessel growth, (b). revascularization following hyperoxia-induced ischemia, or (c). the vasoproliferative phase, indicating a selective function supporting blood vessel survival. We conclude that VEGFR-1 is critical in maintaining the vasculature of the neonatal retina, and that activation of VEGFR-1 by PlGF-1 is a selective strategy for preventing oxygen-induced retinal ischemia without provoking retinal neovascularization.

Inhibition of the mammary carcinoma angiogenic switch in C3(1)/SV40 transgenic mice by a mutated form of human endostatin.

Cancer therapies based on the inhibition of angiogenesis by endostatin have recently been developed. We demonstrate that a mutated form of human endostatin (P125A) can inhibit the angiogenic switch in the C3(1)/Tag mammary cancer model. P125A has a stronger growth-inhibitory effect on endothelial cell proliferation than wild-type endostatin. We characterize the angiogenic switch, which occurs during the transition from preinvasive lesions to invasive carcinoma in this model, and which is accompanied by a significant increase in total protein levels of vascular endothelial growth factor (VEGF) and an invasion of blood vessels. Expression of the VEGF(188) mRNA isoform, however, is suppressed in invasive carcinomas. The VEGF receptors fetal liver kinase-1 (Flk-1) and Fms-like tyrosine kinase-1 (Flt-1) become highly expressed in epithelial tumor and endothelial cells in the mammary carcinomas, suggesting a potential autocrine effect for VEGF on tumor cell growth. Angiopoietin-2 mRNA levels are also increased during tumor progression. CD-31 (platelet-endothelial cell adhesion molecule [PECAM]) staining revealed that blood vessels developed in tumors larger than 1 mm The administration of P125A human endostatin in C3(1)/Tag females resulted in a significant delay in tumor onset, decreased tumor multiplicity and tumor burden and prolonged survival of the animals. Endostatin treatment did not reduce the number of preinvasive lesions, proliferation rates or apoptotic index, compared with controls. However, mRNA levels of a variety of proangiogenic factors (VEGF, VEGF receptors Flk-1 and Flt-1, angiopoietin-2, Tie-1, cadherin-5 and PECAM) were significantly decreased in the endostatin-treated group compared with controls. These results demonstrate that P125A endostatin inhibits the angiogenic switch during mammary gland adenocarcinoma tumor progression in the C3(1)/Tag transgenic model.

Molecular profiling of angiogenesis markers.

The goal of this study was to develop a sensitive, simple, and widely applicable assay to measure copy numbers of specific mRNAs using real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and identify a profile of gene expression closely associated with angiogenesis. We measured a panel of nine potential angiogenesis markers from a mouse transgenic model of prostate adenocarcinoma (TRAMP) and a mouse skin model of vascular endothelial growth factor (VEGF)-driven angiogenesis. In both models, expression of VEGF correlated with expression of mRNAs encoding other angiogenic cytokines (angiopoietin-1 and angiopoietin-2), endothelial cell receptor tyrosine kinases (Flt-1, KDR, Tie-1), and endothelial cell adhesion molecules (VE-cadherin, PECAM-1). Relative to control, in dermis highly stimulated by VEGF, the Ang-2 mRNA transcript numbers increased 35-fold, PECAM-1 and VE-cadherin increased 10-fold, Tie-1 increased 8-fold, KDR and Flt-1 each increased 4-fold, and Ang-1 increased 2-fold. All transcript numbers were correspondingly reduced in skin with less VEGF expression, indicating a relationship of each of these seven markers with VEGF. Thus, this study identifies a highly efficient method for precise quantification of a panel of seven specific mRNAs that correlate with VEGF expression and VEGF-induced neovascularization, and it provides evidence that real-time quantitative RT-PCR offers a highly sensitive strategy for monitoring angiogenesis.