Calcium Ion Mobilization Research Articles

Nanobodies as negative allosteric modulators for human calcium sensing receptor

Human calcium sensing receptor (CaSR) senses calcium ion concentrations in vivo and is an important class of drug targets. Mutations in the receptor can lead to disorders of calcium homeostasis, including hypercalcemia and hypocalcemia. Here, 127 CaSR-targeted nanobodies were generated from camels, and four nanobodies with inhibitory function were further identified. Among these nanobodies, NB32 can effectively inhibit the mobilization of intracellular calcium ions (Ca2+i) and suppress the G12/13 and ERK1/2 signaling pathways downstream of CaSR. Moreover, it enhanced the inhibitory effect of the calcilytics as a negative allosteric modulator (NAM). We determined the structure of complex and found NB32 bound to LB2 (Ligand-binding 2) domain of CaSR to prevent the interaction of LB2 domains of two protomers to stabilize the inactive state of CaSR.

376 CARDIAC CONTRACTILITY MODULATION: A NEW THERAPY FOR HEART FAILURE

Abstract Introduction Cardiac Contractility Modulation is a therapy for chronic Heart Failure patients with EF 25-45% in sinus rhythm with QRS < 130 ms clinically symptomatic (NYHA Class > II) despite optimised medical therapy. It is made possible by the implantation of the OPTIMIZER SMART® device. The CCM works by delivering a non-excitatory high-energy bipolar signal, synchronised with local electrical activity (or cooperating with another pre-existing CIED), in the absolute ventricular refractory period, by means of two actively fixating leads, positioned in the interventricular septum and spaced at least 1-2 cm apart. Purpose Assessment and feedback of the CCM therapy process in patients with dilated cardiomyopathy. Educational update for health professionals. Patient education. Method A Pubmed search correlated with clinical data and field experience was performed. Results Patients with severe Dilated Cardiomyopathy already on optimised medical therapy. The mechanism of action demonstrated a remodelling of the left ventricle with a so-called 'metabolic' therapy. In CCM therapy, electrical stimulation is delivered to the heart muscle during the absolute refractory period. In this phase, electrical signals activate the mobilisation of calcium ions in cardiomyocytes. CCM signals applied during the absolute refractory period cause an increase in cytosolic calcium during systole, resulting in improved cardiac contraction without an increase in oxygen consumption. Conclusions CCM therapy has shown to improve contractility and cardiac performance improving the quality of life of patients undergoing this treatment by reducing symptoms of fatigue and dyspnoea; it has reduced the number of hospitalisations for moderate to severe heart failure. Healthcare professionals recognise The typical ECG of a patient implanted with a CCM device shows a 'spike' in the absolute refractory period of the cardiac cycle: the 'R wave' of the QRS complex without being alarmed. The patient has been trained to be autonomous in charging. OPTIMIZER SMART® is recharged externally with a weekly rechargeable battery through a mini induction charger, rechargeable by itself.

Mammalian STE20-like kinase-1/2 are activated in human platelets stimulated by collagen or thrombin and play a vital role in collagen-activated platelets

IntroductionMammalian ste20-like kinases-1/2 (MST1/2), the core kinases of the Hippo pathway, play critical roles in the biology of hematopoietic cells via noncanonical mechanisms and contributes to megakaryocyte differentiation, polyploidization, and maturation to produce platelets. However, the role of MST1/2 in platelet functions remains unclear. Materials and methodsIn this study, we investigated this topic by determining platelet aggregation and through flow cytometry, ATP release assay, clot retraction assay, and immunoblotting analysis. ResultsWe found that MST1/2 were rapidly phosphorylated and activated upon platelet stimulation by thrombin and collagen. XMU-MP-1, a specific inhibitor of MST1/2, blocks the activation of MST1/2 in platelets. Inhibitor-pretreated platelets showed impaired platelet aggregation and dense-granule secretion mediated by collagen, thrombin, and U46619, whereas ristocetin or ADP mediated platelet aggregation was unaffected by XMU-MP-1. Although platelet-mediated clot retraction was not affected by MST1/2 inhibitors, integrin αIIbβ3 activation was significantly attenuated in XMU-MP-1-treated platelets. Moreover, MST1/2 inhibition significantly attenuated the mobilization of platelet calcium ions and the secretion of α-granules induced by convulxin. ConclusionsThis study is the first to demonstrate that MST1/2 play vital roles in human platelets and contributes to collagen-induced platelet activation and aggregation.

Mechanisms underlying citrinin-induced toxicity via oxidative stress and apoptosis-mediated by mitochondrial-dependent pathway in SH-SY5Y cells

Citrinin (CIT) is a mycotoxin produced as a secondary product by the genera Aspergillus, Penicillium, Monascus, and other strains. CIT has the potential for contaminating animal feed and human food such as maize, wheat, rye, barley, oats, rice, cheese, and sake. Although CIT is primarily known as a nephrotoxic mycotoxin, it also affects other organs, including the liver and bone marrow, and its mechanisms of toxicity have not been clearly elucidated. There is a further lack of studies investigating the potential for CIT-induced neurotoxicity and its mechanisms. In the current study, SH-SY5Y human neuroblastoma cell line was treated with CIT for 24 h to evaluate various toxicological endpoints, such as reactive oxygen species (ROS) production and apoptosis induction. Results indicate that CIT has an IC50 value of 250.90 μM and cell proliferation decreased significantly at 50 and 100 μM CIT concentrations. These same concentrations also caused elevated ROS production (≥34.76%), apoptosis (≥9.43-fold) and calcium ion mobilization (≥36.52%) in the cells. Results show a significant decrease in the mitochondrial membrane potential (≥86.8%). We also found that CIT significantly upregulated the expression of some genes related to oxidative stress and apoptosis, while downregulating others. These results suggest that apoptosis and oxidative stress may be involved in the mechanisms underlying CIT-induced neurotoxicity.

Melatonin attenuates acute and chronic itch in mice: the antioxidant and anti-inflammatory effects of melatonin receptors.

BackgroundItch is a common symptom of skin diseases and significantly reduces patients’ quality of life. Melatonin has anti-inflammatory and antioxidant effects. Our study examined the potential anti-itch effects of melatonin (N-acetyl-5-methoxytryptamine) in mice.MethodsWe detected the effects of melatonin and its receptors on acute and chronic itch by conducting itching behavioral experiments in male C57 mice. Reactive oxygen species (ROS) levels and calcium ion (Ca2+) mobilization during acute itching production were explored using flow cytometry and calcium imaging techniques. Melatonin expression in the serum of the chronic itch model mice was determined by enzyme-linked immunoassays. Hematoxylin and eosin staining show the effects of melatonin on skin thickness in a chronic itch model. Cytokine and chemokine levels were determined by quantitative polymerase chain reaction.ResultsWe discovered that compound 48/80 (C48/80)- and chloroquine (CQ)-induced scratching were significantly decreased by intraperitoneal (i.p), intradermal, and intrathecal administration of melatonin in a dose-dependent manner in mice, and the co-administration of melatonin receptor antagonists abolished the anti-itch effects of i.d melatonin. The incubation of melatonin significantly decreased the intracellular ROS levels induced by C48/80 and CQ in cultured ND7/23 cells from a mouse x rat hybridoma nerve as neuron. Melatonin inhibited intracellular Ca2+ increases induced by CQ (but not C48/80) in cultured dorsal root ganglia (DRG) neurons. Melatonin (50 mg/kg i.p) attenuated imiquimod (IMQ)- or acetone and diethyl ether followed by acetone-ether-water (AEW)-induced chronic itch and epidermal hyperplasia in mice. Finally, melatonin treatment reduced the IMQ-induced expression of ST2 and interleukin-33 (IL-33) or the AEW-induced expression of interleukin 31 (IL-31) and interleukin 31 receptor A (IL-31 RA) in the mice.ConclusionsCollectively, our results indicate that melatonin attenuates acute and chronic itch, possibly via melatonin receptors, and its antioxidant, and anti-inflammatory effects in mice.

ГОМЕОСТАЗ КАЛЬЦИЯ В ООЦИТАХ SUS SCROFA DOMESTICUS ПРИ КУЛЬТИВИРОВАНИИ IN VITRO

The use of the indicator of membrane-bound calcium content (fluorescence intensity of the complex calcium + chlortetracycline + membrane) as a biomarker of the functional state (growing or fully grown oocytes) of donor oocytes in the technology of extracorporeal maturation of female gametes Sus scrofa domesticus is substantiated. The addition of highly dispersed silica nanoparticles with granulosa cells to culture media at in vitro maturation promotes the mobilization of calcium ions from intracellular stores of oocytes that have not finished the growth phase in vivo.

Identification of Potential Human Ryanodine Receptor 1 Agonists and Molecular Mechanisms of Natural Small-Molecule Phenols as Anxiolytics.

Natural small-molecule phenols (NSMPs) possess certain ubiquitous bioactivities including the anxiolytic effect. Ryanodine receptor 1 (RyR1) may be one of the potentially critical pharmacological targets for studying the anxiolytic activity of NSMPs. However, detailed molecular mechanisms of NSMPs have not been fully clarified. This research was intended to identify potent hRyR1 agonists from NSMPs and investigate whether RyR1 plays a role in their anxiolytic effect. Homology modeling and molecular docking analysis were performed using Accelrys Discovery Studio 2.5. The most appropriate concentrations of NSMPs to activate RyR1 were measured using the MTT assay. Fluorescence analyses of the intracellular calcium levels and western blotting analysis were carried out to validate whether NSMPs could regulate the calcium flux to some extent by activating RyR1. The results demonstrated that xanthotoxol and 5-hydroxy-1,4-naphthalenedione can be screened as hit compounds for potential agonists of hRyR1 to exert the anxiolytic effect. In conclusion, NSMPs might be a kind of pharmacological signal carrier, acting on RyR1 as an agonist and resulting in calcium ion mobilization from intracellular calcium ion store.

Enhancement of DPP-IV Inhibitory Activity and GLP-1 Release Through RADA16-assisted Molecular Designed Rapeseed Peptide Nanogels

Abstract Objectives To produce and evaluate the microstructure and rheological behavior of a nanogel of pentapeptide IPQVS (RAP1) and octopeptide ELHQEEPL (RAP2), derived from rapeseed napin, through RADA16-assisted molecular design. In addition, to determine in vitro the dipeptidyl-peptidase IV (DPP-IV) inhibitory properties, glucagon-like peptide-1 (GLP-1) secretion through activation of calcium-sensing receptor (CaSR) and the intracellular calcium ion mobilization and receptor protein cAMP concentration. Methods An innovative nanogel, which loaded the RAP1 and RAP2 into RADA16 scaffold, respectively, was synthesized. The linker of double Gly was used in the connection of RADA16 and the functional oligopeptide region. DPP-IV inhibitory activity was evaluated in the Caco-2 cell monolayer. The microstructure and rheological behavior of RADA16-RAP1 and RADA16-RAP2 were characterized. The GLP-1 secretion through activation of CaSR receptor and the intracellular calcium ion mobilization and cAMP concentration were determined in STC-1 cells. Results DPP-IV inhibitory activity was reduced (more potent) by over 65% in the presence of 250 μM of the two formed nanogels RADA16–GG-IPQVS and RADA16- GG-ELHQEEPL (p < 0.05). The two nanogel peptides had high stability at low temperature or body temperature and high dispersibility in water. Stable β-sheet structures increased by 5.6-fold and 5.2-fold, respectively, than the original oligopeptides, with a self-assembled fibrous morphology. The nanogels RADA16-RAP1 and RADA16-RAP2 might exhibit good rheological properties for potential injectable applications; storage modulus (G’) was 10 times higher than low modulus (G’’). Furthermore, the RAP2 and its RADA16-assisted nanogel peptide at the concentration of 250 μM significantly (p < 0.05) increased the release of GLP-1 by 35.46% through calcium-sensing receptor (CaSR) pathway in the enteroendocrine STC-1 cells. Conclusions The innovated nanogel with the sequence of RADA16-GG-Xn has the possibility of oral and injection to management type 2 diabetes. DPP-IV inhibitory peptides IPQVS and ELHQEEPL can form a nanogel with high water content through the self-assembly gelation of RADA16. They may attach to the brush border of human intestinal epithelial cells to inhibit DPP-IV in the intestinal lumen. Funding Sources USDA-HATCH [grant number 1017440].

Isoprenoid Derivatives of Lysophosphatidylcholines Enhance Insulin and GLP-1 Secretion through Lipid-Binding GPCRs.

Insulin plays a significant role in carbohydrate homeostasis as the blood glucose lowering hormone. Glucose-induced insulin secretion (GSIS) is augmented by glucagon-like peptide (GLP-1), a gastrointestinal peptide released in response to ingesting nutriments. The secretion of insulin and GLP-1 is mediated by the binding of nutrients to G protein-coupled receptors (GPCRs) expressed by pancreatic β-cells and enteroendocrine cells, respectively. Therefore, insulin secretagogues and incretin mimetics currently serve as antidiabetic treatments. This study demonstrates the potency of synthetic isoprenoid derivatives of lysophosphatidylcholines (LPCs) to stimulate GSIS and GLP-1 release. Murine insulinoma cell line (MIN6) and enteroendocrinal L cells (GLUTag) were incubated with LPCs bearing geranic acid (1-GA-LPC), citronellic acid (1-CA-LPC), 3,7-dimethyl-3-vinyloct-6-enoic acid (GERA-LPC), and (E)-3,7,11-trimethyl- 3-vinyldodeca-6,10-dienoic acid (1-FARA-LPC). Respective free terpene acids were also tested for comparison. Besides their insulin- and GLP-1-secreting capabilities, we also investigated the cytotoxicity of tested compounds, the ability to intracellular calcium ion mobilization, and targeted GPCRs involved in maintaining lipid and carbohydrate homeostasis. We observed the high cytotoxicity of 1-GERA-LPC and 1-FARA-LPC in contrast 1-CA-LPC and 1-GA-LPC. Moreover, 1-CA-LPC and 1-GA-LPC demonstrated the stimulatory effect on GSIS and 1-CA-LPC augmented GLP-1 secretion. Insulin and GLP-1 release appeared to be GPR40-, GPR55-, GPR119- and GPR120-dependent.

A mini-review on ion fluxes that regulate NLRP3 inflammasome activation.

The activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome can be induced by a wide spectrum of activators. This is unlikely achieved by the binding of different activators directly to the NLRP3 protein itself, as the activators found so far show different forms of chemical structures. Previous studies have shown that these activators can induce potassium ion (K+) and chloride ion (Cl-) efflux, calcium (Ca2+) and other ion mobilization, mitochondrial dysfunction, and lysosomal disruption, all of which are believed to cause NLRP3 inflammasome activation; how these events are induced by the activators and how they coordinate with each other in inducing the NLRP3 inflammasome activation are not fully understood. Increasing evidence suggests that the coordinated change of intracellular ion concentrations may be a common mechanism for the NLRP3 activation by different activators. In this mini-review, we present a brief summary of the current knowledge about how different ionic flows (including K+, sodium ion, Ca2+, magnesium ion, manganese ion, zinc ion, iron ion, and Cl-) are involved in regulating the NLRP3 inflammasome activation in macrophages.

A novel G protein-coupled receptor for starfish gonadotropic hormone, relaxin-like gonad-stimulating peptide.

Gonadotropic hormones play important regulatory roles in reproduction. Relaxin-like gonad-stimulating peptide (RGP) is a gonadotropin-like hormone in starfish. However, a receptor for RGP remains to be identified. Here, we describe the identification of an authentic receptor for RGP (RGPR) in the starfish, Patiria pectinifera. A binding assay using radioiodinated P. pectinifera RGP (PpeRGP) revealed that RGPR was expressed in ovarian follicle cells. A RGPR candidate was identified by homology-searching of transcriptome data of P. pectinifera follicle cells. Based on the contig sequences, a putative 947-amino acid PpeRGPR was cloned from follicle cells. Like the vertebrate relaxin family peptide receptors (RXFP 1 and 2), PpeRGPR was a G protein-coupled receptor that harbored a low-density lipoprotein-receptor class A motif and leucine-rich repeat sequences in the extracellular domain of the N-terminal region. Sf9 cells transfected with Gαq16-fused PpeRGPR activated calcium ion mobilization in response to PpeRGP, but not to RGP of another starfish Asterias amurensis, in a dose-dependent fashion. These results confirmed the species-specific reactivity of RGP and the cognate receptor. Thus, the present study provides evidence that PpeRGPR is a specific receptor for PpeRGP. To the best of our knowledge, this is the first report on the identification of a receptor for echinoderm RGP.

Enhancement of Intracellular Calcium Ion Mobilization by Moderately but Not Highly Positive Material Surface Charges.

Electrostatic forces at the cell interface affect the nature of cell adhesion and function; but there is still limited knowledge about the impact of positive or negative surface charges on cell-material interactions in regenerative medicine. Titanium surfaces with a variety of zeta potentials between −90 mV and +50 mV were generated by functionalizing them with amino polymers, extracellular matrix proteins/peptide motifs and polyelectrolyte multilayers. A significant enhancement of intracellular calcium mobilization was achieved on surfaces with a moderately positive (+1 to +10 mV) compared with a negative zeta potential (−90 to −3 mV). Dramatic losses of cell activity (membrane integrity, viability, proliferation, calcium mobilization) were observed on surfaces with a highly positive zeta potential (+50 mV). This systematic study indicates that cells do not prefer positive charges in general, merely moderately positive ones. The cell behavior of MG-63s could be correlated with the materials’ zeta potential; but not with water contact angle or surface free energy. Our findings present new insights and provide an essential knowledge for future applications in dental and orthopedic surgery.

Adenosine Receptor Agonists Increase the Inhibition of Platelet Function by P2Y12 Antagonists in a cAMP- and Calcium-Dependent Manner.

We have shown previously that platelet activity can be lowered through the simultaneous inhibition of P2Y12 receptor and activation of adenosine receptors (AR). This work explores this concept by testing the antiplatelet potential of nine AR agonists in combination with P2Y12 receptor antagonists—cangrelor and prasugrel metabolite. A panel of in vitro methods was used to assess platelet viability, P-selectin expression, GPIIb-IIIa activation, fibrinogen binding, calcium ion mobilization, VASP-P level, and cAMP formation, utilizing whole blood or isolated platelets from healthy volunteers. The AR agonists demonstrated anti-platelet effects, but stimulated signaling pathways to varying degrees. AR agonists and P2Y12 antagonists reduced expression of both P-selectin and the activated form of GPIIb-IIIa on platelets; however, the combined systems (AR agonist + P2Y12 antagonist) demonstrated stronger effects. The antiplatelet effects of AR when combined with P2Y12 were more pronounced with regard to exogenous fibrinogen binding and calcium mobilization. The cAMP levels in both resting and ADPactivated platelets were increased by AR agonist treatment, and more so when combined with P2Y12 inhibitor. In conclusion, as AR agonists are fast-acting compounds, the methods detecting early activation events are more suitable for assessing their antiplatelet action. The exogenous fibrinogen binding, calcium mobilisation and cAMP level turned out to be sensitive markers for detecting the inhibition caused by AR agonists alone or in combination with P2Y12 receptor antagonists.

A novel di-peptide Met-Glu from collagen hydrolysates inhibits platelet aggregation and thrombus formation via regulation of Gq-mediated signaling.

Increasing evidence has shown that collagen peptides had various biological activities. In this study, a novel antiplatelet peptide Met-Glu (ME) was separated and identified from silver carp skin by YMC ODS-A C18 separation and ESI-MS/MS analysis. Peptide ME inhibited platelet aggregation and secretion of platelet granules induced by ADP, thrombin and collagen, and significantly attenuated ferric chloride-induced thrombus formation in rats. It did not prolong the bleeding time in mice even at the dose of 300μmol/kg body weight that showed potent anti-thrombosis effects. Additionally, peptide ME targeted at Gq-protein to downregulate the phosphorylation of PLCβ, an important upstream effector of PI3K/Akt and Erk/MAPK signaling to inhibit intracellular calcium ion mobilization. These results suggest that peptide ME inhibited thrombosis in vivo and inhibited Gq-mediated signaling in platelets, indicating the possibility that ME could potentially be developed as a novel therapeutic agent in the prevention and treatment of thrombotic diseases. PRACTICAL APPLICATIONS: Cardiovascular diseases (CVDs) are the leading cause of mortality and morbidity worldwide. The proximal cause of CVDs is intravascular thrombosis formation, which mostly results from platelet activation, aggregation, and granules secretion. Traditional drugs in the prevention of thrombotic disease, such as aspirin and clopidogrel, are still limited for their side effects, especially bleeding complications. Collagen is a natural source for bioactive peptides and our previous study has shown that collagen peptides could inhibit platelet aggregation in vitro. Understanding the mechanism of collagen peptides on regulation of platelet activation and their in vivo anti-thrombosis activities were important for the development of novel-specific medical food in the prevention of thrombotic diseases.

Analysis of Platelet Aggregation, Secretion, Integrin Activation, and Calcium Release Stimulated by Different Agonists in Native Americans

Arterial thrombosis results from obstructive vascular blood clots that are largely initiated by platelet activation. As such, individuals whose platelets react more robustly to chemical signals generated by vascular damage or dysfunction are at a higher risk for morbidity and mortality caused by thrombosis. As a population, Native Americans have higher rates of obstructive arterial diseases, and previous research suggests African American populations have higher overall platelet reactivity that positively correlates to their higher risk for cardiovascular disease. Therefore, we set out to measure platelet reactivity in Native Americans and identify potential genetic alleles that correlate to the elevations in responsivity. Five platelet agonists were utilized to simulate vascular damage signals, followed by measurements of markers indicative of subsequent platelet activation: aggregation, secretion, integrin activation, and calcium ion mobilization. Preliminary results from 17 subjects showed that Native American platelets more rapidly aggregate in response to several agonists when compared with Caucasians, whereas other measures of platelet function are largely similar between the groups. These results suggest that Native Americans have more sensitive platelet aggregation responses, which could result in a higher risk for occlusive arterial diseases. Complete genomic sequences were also obtained from the highest responders for use in identifying alleles that correlate to this response.Support or Funding InformationInstitutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103443

Enhancement by Hydrogen Peroxide of Calcium Signals in Endothelial Cells Induced by 5-HT1B and 5-HT2B Receptor Agonists.

Hydrogen peroxide, formed in the endothelium, acts as a factor contributing to the relaxation of blood vessels. The reason for this vasodilatory effect could be modulation by H2O2 of calcium metabolism, since mobilization of calcium ions in endothelial cells is a trigger of endothelium-dependent relaxation. The aim of this work was to investigate the influence of H2O2 on the effects of Ca2+-mobilizing agonists in human umbilical vein endothelial cells (HUVEC). We have found that H2O2 in concentration range 10-100 μM increases the rise of [Ca2+]i induced by 5-hydroxytryptamine (5-HT) and carbachol and does not affect the calcium signals of ATP, agonist of type 1 protease-activated receptor SFLLRN, histamine and bradykinin. Using specific agonists of 5-HT1B and 5-HT2B receptors CGS12066B and BW723C86, we have demonstrated that H2O2 potentiates the effects mediated by these types of 5-HT receptors. Potentiation of the effect of BW723C86 can be produced by the induction of endogenous oxidative stress in HUVEC. We have shown that the activation of 5-HT2B receptor by BW723C86 causes production of reactive oxygen species (ROS). Inhibitor of NADPH oxidases VAS2870 suppressed formation of ROS and partially inhibited [Ca2+]i rise induced by BW723C86. Thus, it can be assumed that vasorelaxation induced by endogenous H2O2 in endothelial cells partially occurs due to the potentiation of the agonist-induced calcium signaling.

Phenotypic stability of the human MG-63 osteoblastic cell line at different passages.

One of the most popular cell lines in osteogenesis studies is the human osteoblastic line MG-63. For cell biological investigation, it is important that the cells remain stable in their phenotype over several passages in cell culture. MG-63 cells can be used to provide fundamental insights into cell--material interaction. The aim of this study is to present a systematic characterization of the physiological behavior of MG-63 cells in the range of passages 5-30. Significant cell physiology processes during the first 24 h, including cell morphology, availability of adhesion receptors, cell cycle phases, as well as the expression of the signaling proteins Akt, GSK3a/b, IkB-α, ERK1/2, p38-MAPK, and intracellular calcium ion mobilization, remained stable over the entire range of passages P5-P30. Due to these stable characteristics in a wide range of cell culture passages, MG-63 cells can be considered as a suitable in vitro model to analyze the biocompatibility and biofunctionality of implant materials.

Gintonin modulates platelet function and inhibits thrombus formation via impaired glycoprotein VI signaling.

Panax ginseng (P. ginseng), one of the most valuable medicinal plants, is known for its healing and immunobooster properties and has been widely used in folk medicine against cardiovascular diseases, including stroke and heart attack. In this study, we explored the anti-platelet activity of gintonin (a recently discovered non-saponin fraction of ginseng) against agonist-induced platelet activation. In vitro effects of gintonin on agonist-induced human and rat platelet aggregation, granule secretion, integrin αIIbβ3 activation, and intracellular calcium ion ([Ca2+]i) mobilization were examined. Western blot analysis and immunoprecipitation techniques were used to estimate the expression of mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) and interaction of glycoprotein VI (GPVI) signaling pathway molecules such as Src family kinases (SFK), tyrosine kinase Syk, and PLCγ2. In vivo effects were studied using acute pulmonary thromboembolism model in mice. Gintonin remarkably inhibited collagen-induced platelet aggregation and suppressed granule secretion, [Ca2+]i mobilization, and fibrinogen binding to integrin αIIbβ3 in a dose-dependent manner and clot retraction. Gintonin attenuated the activation of MAPK molecules and PI3K/Akt pathway. It also inhibited SFK, Syk, and PLCγ2 activation and protected mice from thrombosis. Gintonin inhibited agonist-induced platelet activation and thrombus formation through impairment in GPVI signaling molecules, including activation of SFK, Syk, PLCγ2, MAPK, and PI3K/Akt; suggesting its therapeutic potential against platelet related CVD.

Enhanced calcium ion mobilization in osteoblasts on amino group containing plasma polymer nanolayer

BackgroundBiomaterial modifications—chemical and topographical—are of particular importance for the integration of materials in biosystems. Cells are known to sense these biomaterial characteristics, but it has remained unclear which physiological processes bio modifications trigger. Hence, the question arises of whether the dynamic of intracellular calcium ions is important for the characterization of the cell–material interaction. In our prior research we could demonstrate that a defined geometrical surface topography affects the cell physiology; this was finally detectable in a reduced intracellular calcium mobilization after the addition of adenosine triphosphate (ATP).ResultsThis new contribution examines the cell physiology of human osteoblasts concerning the relative cell viability and the calcium ion dynamic on different chemical modifications of silicon–titanium (Ti) substrates. Chemical modifications comprising the coating of Ti surfaces with a plasma polymerized allylamine (PPAAm)-layer or with a thin layer of collagen type-I were compared with a bare Ti substrate as well as tissue culture plastic. For this purpose, the human osteoblasts (MG-63 and primary osteoblasts) were seeded onto the surfaces for 24 h. The relative cell viability was determined by colorimetric measurements of the cell metabolism and relativized to the density of cells quantified using crystal violet staining. The calcium ion dynamic of osteoblasts was evaluated by the calcium imaging analysis of fluo-3 stained vital cells using a confocal laser scanning microscope. The positively charged nano PPAAm-layer resulted in enhanced intracellular calcium ion mobilization after ATP-stimulus and cell viability. This study underlines the importance of the calcium signaling for the manifestation of the cell physiology.ConclusionsOur current work provides new insights into the intracellular calcium dynamic caused by diverse chemical surface compositions. The calcium ion dynamic appears to be a sensitive parameter for the cell physiology and, thus, may represent a useful approach for evaluating a new biomaterial. In this regard, reliable in vitro-tests of cell behavior at the interface to a material are crucial steps in securing the success of a new biomaterial in medicine.

Eisenia bicyclis (brown alga) modulates platelet function and inhibits thrombus formation via impaired P2Y12 receptor signaling pathway

Background and purposeSea weeds have been used since ancient times in Asian countries, especially in Korea, Japan, and China, as both edible sea vegetables and traditional medicinal tonics due to their health benefits. Eisenia bicyclis has been studied for anti-allergic and anti-cancer effects; however, its effects on the cardiovascular system, especially on platelet function, are yet to be explored. Therefore, we examined the effect of E. bicyclis on platelet function. Study design and methodsE. bicyclis extract (EBE) was prepared and in vitro effects on ADP-induced platelet aggregation, granule secretion, intracellular calcium ion ([Ca2+]i) mobilization, fibrinogen binding to integrin αIIbβ3 and clot retraction were evaluated. Phosphorylation levels of MAPK signaling molecules and P2Y12 receptor downstream signaling pathway components were studied. In vivo effects were studied using an arteriovenous (AV) shunt model. ResultsEBE markedly inhibited in vitro ADP-induced platelet aggregation, granule secretion (ATP release and P-selectin expression), [Ca2+]i mobilization, fibrinogen binding to integrin αIIbβ3, and clot retraction; attenuated MAPK pathway activation; and inhibited phosphorylation of PI3K/Akt, PLCγ2, and Src. The extract significantly inhibited in vivo thrombus weight in an AV shunt model. ConclusionE. bicyclis inhibits agonist-induced platelet activation and thrombus formation through modulation of the P2Y12 receptor downstream signaling pathway, suggesting its therapeutic potential in ethnomedicinal applications as an anti-platelet and anti-thrombotic compound to prevent cardiovascular diseases.