Cytosolic calcium microdomains by arachidonic acid and nitric oxide in endothelial cells

Intracellular calcium signals activated by growth factors in endothelial cells during angiogenesis regulate cytosolic and nuclear events involved in survival, proliferation and motility. Among the intracellular messengers released after proangiogenic stimulation (bFGF, VEGF), arachidonic acid (AA), nitric oxide (NO) and their metabolites play a key role and their effects are strictly related to calcium homeostasis. Recently, we showed that AA and NO are able to stimulate the opening of store-independent calcium-permeable channels in the plasmamembrane of bovine aortic endothelial cells (BAECs). Here, we studied the intracellular spatiotemporal dynamics of AA- and NO-induced calcium increases following store-independent calcium entry from extracellular medium. Using confocal calcium imaging, we show that calcium entry is preferentially restricted to peripheral cytosolic microdomains and does not necessarily invade the nuclear region. These results support the existence of local mitogen-activated calcium signals. Several factors could account for this spatial restriction, including the geometry of the cells and the clusterization of calcium channels and other signalling molecules. Intracellular calcium fingerprints could contribute to the specificity of endothelial cell responses to angiogenic factors.     

A systems approach to investigate GPCR-mediated Ras signaling network in chemoattractant sensing

A GPCR-mediated signaling network enables a chemotactic cell to generate adaptative Ras signaling in response to a large range of concentrations of a chemoattractant. To explore potential regulatory mechanisms of GPCR-controlled Ras signaling in chemosensing, we applied a software package, Simmune, to construct detailed spatiotemporal models simulating responses of the cAR1-mediated Ras signaling network. We first determined the dynamics of G-protein activation and Ras signaling in Dictyostelium cells in response to cAMP stimulations using live-cell imaging and then constructed computation models by incorporating potential mechanisms. Using simulations, we validated the dynamics of signaling events and predicted the dynamic profiles of those events in the cAR1-mediated Ras signaling networks with defective Ras inhibitory mechanisms, such as without RasGAP, with RasGAP overexpression, or with RasGAP hyperactivation. We describe a method of using Simmune to construct spatiotemporal models of a signaling network and run computational simulations without writing mathematical equations. This approach will help biologists to develop and analyze computational models that parallel live-cell experiments.     

Adrenomedullin stimulates angiogenic response in cultured human vascular endothelial cells: involvement of the vascular endothelial growth factor receptor 2

In recent years, evidence has accumulated that many endogenous peptides play an important regulatory role in angiogenesis by modulating endothelial cell behavior. Adrenomedullin (AM), one such factor, was previously shown to exert a clearcut proangiogenic effect in vitro when tested on specialized human endothelial cells, such as HUVECs and immortalized endothelial cell lines. In the present study we used normal adult vascular endothelial cells isolated from human saphenous vein to analyze in vitro the role of AM, related to both early (increased cell proliferation) and late (differentiation and self-organization into capillary-like structures) angiogenic events and their relationship with the vascular endothelial growth factor (VEGF) signaling cascade. The results indicated that also in this endothelial cell phenotype AM promoted cell proliferation and differentiation into cord-like structures. These actions resulted specific and were mediated by the binding of AM to its AM1 (CRLR/RAMP2) receptor. Neither the administration of a VEGF receptor 2 (VEGFR-2) antagonist nor the downregulation of VEGF production by gene silencing were able to suppress the proangiogenic effect of AM. However, when the experiments were performed in the presence of SU5416 (a selective inhibitor of the VEGFR-2 receptor at the level of the intra-cellular tyrosine kinase domain) the proangiogenic effect of AM was abolished. This result suggests that in vascular endothelial cells the binding of AM to its AM1 receptor could trigger a transactivation of the VEGFR-2 receptor, leading to a signaling cascade inducing proangiogenic events in the cells.     

Notch和Wnt信号通路在血管形成中的协同调控作用

Notch和Wnt信号通路能够调控细胞的分化、增殖、迁移和粘附等多种行为,在胚胎发育、干细胞分化及肿瘤生长等方面发挥多样性的调控作用.血管形成过程中的典型事件包括尖端细胞(tipcell)和柄细胞(stalkcell)分化、柄细胞增殖、内皮细胞迁移和粘附、血管重塑以及动静脉分化等.本文对Notch和Wnt信号通路在血管形成不同阶段的功能作一综述,以期描述Notch和Wnt是怎样在分子水平上协同作用进而调控血管的形成.从两条信号通路的分子水平及复杂信号网络中众多成员协调作用的角度了解血管形成的机制,对于调整肿瘤等涉及血管形成的相关疾病的治疗策略具有一定意义.     

Elevation of intracellular calcium levels in neurons by nicotinic acetylcholine receptors

The recognition that intracellular free calcium serves as a ubiquitous intracellular signal has motivated efforts to elucidate mechanisms by which cells regulate calcium influx. One route of entry that may offer both spatial and temporal fine resolution for altering calcium levels is that provided by cation-permeable, ligand-gated ion channels. Biophysical measurements as well as calcium imaging techniques demonstrate that neuronal nicotinic acetylcholine receptors as a class have a high relative permeability to calcium; some subtypes equal or exceed all other known receptors in this respect. Activation of nicotinic receptors on neurons can produce substantial increases in intracellular calcium levels by direct passage of calcium through the receptor channel. When multiple classes of nicotinic receptors are expressed by the same neuron, each appears capable of increasing calcium in the cell but may differ with respect to location, temporal response, agonist sensitivity, or regulation in achieving it. As a result, nicotinic receptors must be considered strong candidates for signaling molecules through which neurons regulate a diverse array of cellular events.     

Skeletal muscle VEGF gradients in peripheral arterial disease: simulations of rest and exercise

VEGF is a key promoter of angiogenesis and a major target of proangiogenic therapy for peripheral arterial disease (PAD). Greater understanding of VEGF angiogenic signaling and guidance by gradients for new capillaries will aid in developing new proangiogenic therapies and improving existing treatments. However, in vivo measurements of VEGF concentration gradients at the cell scale are currently impossible. We have developed a computational model to quantify VEGF distribution in extensor digitorum longus skeletal muscle using measurements of VEGF, VEGF receptor (VEGFR), and neuropilin-1 (NRP1) expression in an experimental model of rat PAD. VEGF is secreted by myocytes, diffuses through and interacts with extracellular matrix and basement membranes, and binds VEGFRs and NRP1 on endothelial cell surfaces of blood vessels. We simulate the effects of increased NRP1 expression and of therapeutic exercise training on VEGF gradients, receptor signaling, and angiogenesis. Our study predicts that angiogenic therapy for PAD may be achieved not only through VEGF upregulation but also through modulation of VEGFRs and NRP1. We predict that expression of 10(4) NRP1/cell can increase VEGF binding to receptors by 1.7-fold (vs. no NRP1); in nonexercise-trained muscle with PAD, angiogenesis is hindered due to limited VEGF upregulation, signaling, and gradients; in exercise-trained muscle, VEGF signaling is enhanced by upregulation of VEGFRs and NRP1, and VEGF signaling is strongest within the first week of exercise therapy; and hypoxia-induced VEGF release is important to direct angiogenesis towards unperfused tissue.     

Mechanistic systems models of cell signaling networks: a case study of myocyte adrenergic regulation

Signal transduction networks coordinate a wide variety of cellular functions, including gene expression, metabolism, and cell fate processes. Understanding biological networks quantitatively is a major challenge to post-genomic biology, and mechanistic systems models will be crucial for this task. Here, we review approaches towards developing mechanistic systems models of established cell signaling networks. The ability of mechanistic system models to generate testable biological hypotheses and experimental strategies is discussed. As a case study of model development and analysis, we examined the functional roles of phospholamban, the L-type calcium channel, the ryanodine receptor, and troponin I phosphorylation upon β-adrenergic stimulation in the rat ventricular myocyte. Model analysis revealed that while protein kinase A-mediated phosphorylation of the ryanodine receptor greatly increases its calcium sensitivity, calcium autoregulation may adapt quickly by negating potential increases in contractility. Systematic combinations of in silico perturbations supported the conclusion that phospholamban phosphoregulation is the primary mechanism for increased sarcoplasmic reticulum load and calcium relaxation rate during β-adrenergic stimulation, while both phospholamban and the L-type calcium channel contribute to increased systolic calcium. Combined with detailed experimental studies, mechanistic systems models will be valuable for developing a quantitative understanding of cell signaling networks.     

Application of a systems approach to study developmental gene regulation

All cells in a multicellular organism contain the same genome, yet different cell types express different sets of genes. Recent advances in high throughput genomic technologies have opened up new opportunities to understand the gene regulatory network in diverse cell types in a genome-wide manner. Here, I discuss recent advances in experimental and computational approaches for the study of gene regulation in embryonic development from a systems perspective. This review is written for computational biologists who have an interest in studying developmental gene regulation through integrative analysis of gene expression, chromatin landscape, and signaling pathways. I highlight the utility of publicly available data and tools, as well as some common analysis approaches.     

Heme oxygenase-1 and cardiovascular disease

Heme oxygenase (HO)-1 is the inducible isoform of the first and rate-controlling enzyme of heme degradation. HO-1 is up-regulated by a host of oxidative stress stimuli and has potent cytoprotective and anti-inflammatory functions via decreasing tissue levels of the prooxidant heme along with production of bilirubin and the signaling gas carbon monoxide. This review deals with recent findings that highlight the emerging significance of HO-1 in cardiovascular disease. Evidence is presented on how heme and various oxidative stress stimuli may cause endothelial cell dysfunction and how HO-1 may counteract the detrimental effects of oxidative stress in the endothelium. Recent advances in the understanding of the role of endothelial HO-1 for the regulation of the inflammatory response are summarized, including the modulation of leukocyte recruitment and transmigration through the endothelial barrier. Furthermore, experimental evidence from various cell culture and animal models is discussed which suggests an association of HO-1 with the complex sequence of events that cause atherosclerosis. In the second part of the review we present potential strategies that apply HO-1 as a therapeutic target in the treatment of cardiovascular disease. Specific inducers of HO-activity which may ultimately lead to the development of clinically relevant pharmacological applications are introduced.     

Actinomyosin Contraction,Phosphorylation of VE-Cadherin,and Actin Remodeling Enable Melanoma-Induced Endothelial Cell-Cell Junction Disassembly

During melanoma cell extravasation through the vascular endothelium, melanoma cells interact with endothelial cells through secretion of cytokines and by adhesion between proteins displayed on opposing cell surfaces. How these tumor cell associated signals together regulate the dynamics of intracellular signaling pathways within endothelial cells leading to endothelial cell-cell junction disruption is not well understood. Here, we used a combination of experimental and computational approaches to examine the individual and combined effects of activation of the vascular cell adhesion molecule (VCAM)-1, interleukin (IL)-8, and IL-1β signaling pathways on the integrity of vascular junctions. Our simulations predict a multifaceted interplay of signaling resulting from individual activation of VCAM-1, IL-8 and IL-1β pathways that is neither synergistic nor additive compared to all inputs turned on simultaneously. Furthermore, we show that the levels of phosphorylated proteins associated with actinomyosin contractility and junction disassembly peak prior to those related to actin remodeling. The results of this work provide insight into the dynamics of tumor-mediated endothelial junction disassembly and suggest that targeting proteins downstream of several interaction pathways may be the most effective therapeutic approach to reduce melanoma extravasation.     

钙信号基本单位和特征的研究进展

细胞内存在多种不同的Ｃａ＾２＋信号基本单位,这些Ｃａ＾２＋信号基本单位依赖于刺激浓度的等级体系组织。低水平的刺激激活单通道开放,产生Ｃａ＾２＋脉冲或Ｃａ＾２＋夸克;在等组织水平刺激则产生喷烟和火花,似乎与一小簇通道的激活有关;高浓度刺激时,Ｃａ＾２＋信号基本单位协同产生球形Ｃａ＾２＋波。这些Ｃａ＾２＋基本单位既本现了钙释放单位（Ｃａ＾２＋ｒｅｌｅａｓｅ ｕｎｉｔ）的特征,又导致Ｃａ＾２＋信号传播在     

A Multiscale Hybrid Model for Pro-angiogenic Calcium Signals in a Vascular Endothelial Cell

Cytosolic calcium machinery is one of the principal signaling mechanisms by which endothelial cells (ECs) respond to external stimuli during several biological processes, including vascular progression in both physiological and pathological conditions. Low concentrations of angiogenic factors (such as VEGF) activate in fact complex pathways involving, among others, second messengers arachidonic acid (AA) and nitric oxide (NO), which in turn control the activity of plasma membrane calcium channels. The subsequent increase in the intracellular level of the ion regulates fundamental biophysical properties of ECs (such as elasticity, intrinsic motility, and chemical strength), enhancing their migratory capacity. Previously, a number of continuous models have represented cytosolic calcium dynamics, while EC migration in angiogenesis has been separately approached with discrete, lattice-based techniques. These two components are here integrated and interfaced to provide a multiscale and hybrid Cellular Potts Model (CPM), where the phenomenology of a motile EC is realistically mediated by its calcium-dependent subcellular events. The model, based on a realistic 3-D cell morphology with a nuclear and a cytosolic region, is set with known biochemical and electrophysiological data. In particular, the resulting simulations are able to reproduce and describe the polarization process, typical of stimulated vascular cells, in various experimental conditions. Moreover, by analyzing the mutual interactions between multilevel biochemical and biomechanical aspects, our study investigates ways to inhibit cell migration: such strategies have in fact the potential to result in pharmacological interventions useful to disrupt malignant vascular progression.     

Plant calcium signaling and monitoring: pros and cons and recent experimental approaches

Summary This review focusses on Ca²⁺-mediated plant cell signaling and optical methods for in vivo [Ca²⁺] monitoring and imaging in plants. The cytosolic free calcium concentration has long been considered the central cellular key in plants. However, more and more data are turning up that critically question this view. Conflicting arguments show that there are still many open questions. One conclusion is that the cytosolic free Ca²⁺ concentration is just one of many cellular network parameters orchestrating complex cellular signaling. Novel experimental strategies which unveil interference of cellular parameters and communication of transduction pathways are required to understand this network. To date only optical methods are able to provide both kinetic and spatial information about cellular key parameters simultaneously. Focussing on calcium there are currently three classes of calcium indicators employed (i.e., chemical fluorescent dyes, luminescent indicators, and green-fluorescent-protein-based indicators). Properties and capabilities as well as advantages and disadvantages of these indicators when used in plant systems are discussed. Finally, general experimental strategies are mentioned which are able to answer open questions raised here.Abbreviations CTZ coelenterazine - GFP green-fluorescent protein - FRET fluorescence resonance energy transfer - [Ca²⁺] calcium ion concentration - CaM calmodulin - CDPKs calmodulindomain protein kinases - IP₃ inositol 1,4,5-trisphosphate     

Sphingosine 1-phosphate activates Akt, nitric oxide production, and chemotaxis through a Gi protein/phosphoinositide 3-kinase pathway in endothelial cells

Sphingosine 1-phosphate (SPP) binds to members of the endothelial differentiation gene family (EDG) of receptors and leads to diverse signaling events including cell survival, growth, migration and differentiation. However, the mechanisms of how SPP activates these proangiogenic pathways are poorly understood. Here we show that SPP signals through the EDG-1 receptor to the heterotrimeric G protein G(i), leading to activation of the serine/threonine kinase Akt and phosphorylation of the Akt substrate, endothelial nitric-oxide synthase (eNOS). Inhibition of G(i) signaling, and phosphoinositide 3-kinase (PI 3-kinase) activity resulted in a decrease in SPP-induced endothelial cell chemotaxis. SPP also stimulates eNOS phosphorylation and NO release and these effects are also attenuated by inhibition of G(i) signaling, PI 3-kinase, and Akt. However, inhibition of NO production did not influence SPP-induced chemotaxis but effectively blocked the chemotactic actions of vascular endothelial growth factor. Thus, SPP signals through G(i) and PI 3-kinase leading to Akt activation and eNOS phosphorylation.     

Click chemistry and optogenetic approaches to visualize and manipulate phosphatidic acid signaling

The simple structure of phosphatidic acid (PA) belies its complex biological functions as both a key phospholipid biosynthetic intermediate and a potent signaling molecule. In the latter role, PA controls processes including vesicle trafficking, actin dynamics, cell growth, and migration. However, experimental methods to decode the pleiotropy of PA are sorely lacking. Because PA metabolism and trafficking are rapid, approaches to accurately visualize and manipulate its levels require high spatiotemporal precision. Here, we describe recent efforts to create a suite of chemical tools that enable imaging and perturbation of PA signaling. First, we describe techniques to visualize PA production by phospholipase D (PLD) enzymes, which are major producers of PA, called Imaging Phospholipase D Activity with Clickable Alcohols via Transphosphatidylation (IMPACT). IMPACT harnesses the ability of endogenous PLD enzymes to accept bioorthogonally tagged alcohols in transphosphatidylation reactions to generate functionalized reporter lipids that are subsequently fluorescently tagged via click chemistry. Second, we describe two light-controlled approaches for precisely manipulating PA signaling. Optogenetic PLDs use light-mediated heterodimerization to recruit a bacterial PLD to desired organelle membranes, and photoswitchable PA analogs contain azobenzene photoswitches in their acyl tails, enabling molecular shape and bioactivity to be controlled by light. We highlight select applications of these tools for studying GPCR–G_q signaling, discovering regulators of PLD signaling, tracking intracellular lipid transport pathways, and elucidating new oncogenic signaling roles for PA. We envision that these chemical tools hold promise for revealing many new insights into lipid signaling pathways.     

Rearrangement of the endoplasmic reticulum and calcium transient formation: the computational approach

Experiments affecting calcium signaling often lead to changes in the calcium transient height. The present work is designed to approach this effect theoretically. Use of computational model let us to follow results of precisely designed changes in the endoplasmic reticulum distribution as a possible cause of cytoplasmic free calcium ion level. Obtained results suggest that indeed, rearrangement of the endoplasmic reticulum elements may be responsible for modulation of calcium signal's strength. We have also noticed that even if the endoplasmic reticulum concentration levels are local, the resulting changes in free calcium concentration are global and evenly distributed throughout the cell. The used mathematical method proved to be a powerful tool which made us understand the chemical dynamics of nonequilibrium processes of calcium transient formation. Presented data show how Ca2+ signal resulting from IP3 provoked release of calcium from the endoplasmic reticulum may depend on the cytoskeleton structure.     

c-Myc Oncoprotein: Cell Cycle-Related Events and New Therapeutic Challenges in Cancer and Cardiovascular Diseases

Advanced stages of both cancer and atherosclerosis are characterized by a local increase in tissue mass that may be hard to control. This increase in tissue mass can be attributed to oxidation-sensitive modification of cell cycle-related events, including cellular proliferation, differentiation, and apoptosis, which could be secondary to alteration in the activity of tumor suppressor gene and oncogene products. The oncogene c-Myc has classically been considered to be involved in carcinogenesis and has more recently been implicated in both endothelial dysfunction and atherogenesis as well. Consequently, inhibition of c-Myc-dependent signaling has become a novel therapeutic opportunity and challenge in atherosclerosis and other cardiovascular diseases.

Antioxidant strategies, RNA synthesis inhibitors such as mithramycin, and gene therapeutic approaches with antisense oligonucleotides against c-Myc are some of the promising strategies. In general, the increased biologic understanding of the participation of cell cycle events and targeting these events may enable to attenuate or prevent some of the complications of vascular and neoplastic diseases.