Abstract 210: Biosynthesis of D-series Resolvin by Isolated Vascular Cells and Tissues

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Sevan R Komshian ◽  
Anuran Chatterjee ◽  
Bian Wu ◽  
Giorgio Mottola ◽  
Mian Chen ◽  
...  
Keyword(s):  
Vascular Tissue ◽  
De Novo ◽  
Culture Media ◽  
Primary Cultures ◽  
Rabbit Aorta ◽  
Inflammatory Cells ◽  
Local Source ◽  
Vascular Cells ◽  
Resolvin D1

Introduction: Resolvin-D1 (RvD1) and other specialized pro-resolving lipid mediators (SPM) are synthesized in-vivo from docosahexaenoic acid (DHA) through transcellular pathways involving leukocytes. We investigated if vascular tissues, in the absence of inflammatory cells, can contribute to the local production of SPM. Methods: Primary cultures of human saphenous vein endothelial (EC) and smooth muscle (SMC) cells were supplemented with DHA in cell culture media (10% serum) for 4h-24h. Freshly harvested rabbit aorta was incubated intact or following gentle EC denudation in medium with or without DHA for 48h. RvD1 levels were quantified by ELISA, and lipoxygenase (LO) expression by western blotting. Results: In the absence of DHA supplementation, EC and SMC produced undetectable levels of RvD1. DHA treatment produced a dose and time-dependent increase in RvD1 production by EC and SMC (10.1 ±1.0 pg, 7.4 ±0.2 pg respectively; 1000nM DHA; 24h; Fig A, B). 5-LO expression was demonstrated in both cell types, however DHA induced increased 5-LO expression in EC (Fig C) but not in SMC. DHA-treated intact rabbit aorta segments produced 0.24±0.05 pg RvD1/mg tissue versus 0.13±0.01 pg RvD1/mg tissue in media alone. Moreover, EC-denuded aortas produced significantly less RvD1 (Fig D). Conclusions: Human vascular cells and rabbit vascular tissue can biosynthesize RvD1 de novo from its precursor DHA, signifying a potentially important local source of SPM in the vasculature.

Kinin receptors in experimental inflammation

1980 ◽  
Vol 58 (5) ◽  
pp. 536-542 ◽  
Author(s):  
F. Marceau ◽  
J. Barabé ◽  
S. St-Pierre ◽  
D. Regoli
Keyword(s):  
Urinary Bladder ◽  
De Novo ◽  
Rabbit Aorta ◽  
Initial Response ◽  
Rat Urinary Bladder ◽  
Chemical Mediation ◽  
Intravesical Injection ◽  
Triton X

The contractile response of the rat isolated urinary bladder to kinins is mediated by receptors of the B1 and of the B2 types, as this preparation responds to des-Arg9-bradykinin (des-Arg9-BK), a fairly selective stimulant of receptor B1 and to [Tyr(Me)8]-BK, a potent agonist on receptor B2. Des-Arg10-[Leu9]-kallidin, a specific and competitive antagonist of the action of kinins on receptor B1, has been found to block the effect of des-Arg9-BK in concentrations similar to those required in the rabbit aorta; therefore, the B1 receptor of the rat urinary bladder is analogous to that of the rabbit vascular tissue.The response of the rat urinary bladder to des-Arg9-BK increases progressively from near null level during the incubation in vitro and can be abolished by cycloheximide; this suggests that receptor B1 of the rat urinary bladder is formed de novo.The inflammation of the bladder induced by intravesical injection of the detergent Triton X-100 enhances the initial response to des-Arg9-BK without modifying the response to other agents. The B1 receptor is formed in vivo in the rat urinary bladder submitted to the Triton X-100 treatment but not in the control untreated organ. The local de novo synthesis of B1 receptors for kinins that follows a noxious stimulus is proposed as a possible mechanism implicated in the chemical mediation of the inflammatory process.

Atomic Force and Confocal Microscopic Studies of Collagen-Cell-Based Scaffolds for Vascular Tissue Engineering

2006 ◽  
Vol 15-17 ◽  
pp. 83-88 ◽  
Author(s):  
Luca Amadori ◽  
Navneeta Rajan ◽  
Simone Vesentini ◽  
D. Mantovani
Keyword(s):  
Tissue Engineering ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Tissue ◽  
Collagen Gel ◽  
Muscle Cells ◽  
Vascular Tissue Engineering ◽  
Vascular Cells ◽  
Atomic Force

Collagen is the most used naturally occurring scaffold material. It’s a structural protein ubiquitous among mammalian. The ability of collagen type I to host different cell phenotype in vitro and its low antigenecity in vivo are well known. However, the principal drawback of collagenbased materials consists in their low mechanical properties. For vascular tissue engineering this represents a major limit, as the aim is to mimic the structure of a native vessel, which is known to be resistant and viscoelastic. Moreover, vascular cells are known to be susceptible in vivo to reorganize the matrix in which they proliferate. Therefore, the aim of this project is to study the micro structural organization of collagen-based scaffolds, and to assess the interactions between collagen and smooth muscle cells during regeneration. This knowledge will then allow the development of appropriate strategies to tailor the microstructure of the scaffold and its properties. Smooth muscle cells (SMCs) were selected to study the interactions between cells and matrix during the proliferation. Atomic Force Microscopy (AFM) in dry state in tapping mode and Confocal Laser Scanning Microscopy (CLSM) in reflection mode were used to investigate the microstructure of the scaffold. For the former technique cells were seeded on top of the collagen gel after jellification, while for the latter, cells were embedded into the collagen gel and stained with Rhodamine. The contact points between matrix and cells were investigated, as well as the capacity of vascular cells to induce a structural reorganization of collagen fibrils in the scaffold.

scholarly journals MCP-1/CCR2B-dependent loop upregulates MUC5AC and MUC5B in human airway epithelium

2011 ◽  
Vol 300 (2) ◽  
pp. L204-L215 ◽  
Author(s):  
Maria E. Monzon ◽  
Rosanna Malbrán Forteza ◽  
S. Marina Casalino-Matsuda
Keyword(s):  
Airway Epithelium ◽  
Primary Cultures ◽  
Airflow Obstruction ◽  
Inflammatory Cells ◽  
Chronic Obstructive ◽  
Cell Hyperplasia ◽  
Epithelial Surface ◽  
Initial Exposure ◽  
Mucus Hypersecretion

Cigarette smoke represents a major risk factor for the development of chronic obstructive pulmonary disease (COPD), a respiratory condition associated with airflow obstruction, mucus hypersecretion, chronic inflammation, and upregulation of inflammatory mediators such as the monocyte chemotactic protein-1 (MCP-1). MCP-1 through its receptor CCR2 induces chemotaxis and activates 44/42MAPK, a kinase known to play a key role in mucin regulation in bronchial epithelium. In the present study we used differentiated primary cultures of normal human bronchial epithelial (NHBE) cells to test whether MCP-1 through its receptor CCR2 induces mucin upregulation. We have provided evidence that NHBE cells release MCP-1 to the epithelial surface and express the CCR2B isoform of the receptor mainly at the apical pole. In addition, we found that MCP-1 has a novel function in airway epithelium, increasing the two major airway mucins MUC5AC and MUC5B, an effect mediated, at least in part, by a cascade of events initiated by interaction of its receptor CCR2B with Gq subunits in caveolae, followed by PLCβ, PKC, and 44/42MAPK activation. We also have shown that MCP-1 is able to induce its own expression using the same receptor but through a different pathway that involves RhoA GTPase. Furthermore, we found that a single exposure to MCP-1 is enough to induce MCP-1 secretion and sustained mucin upregulation up to 7 days after initial exposure, an effect mediated by CCR2B as confirmed using short hairpin RNA. These results agree with our data in smoker's airway epithelium, where CCR2B is present in MUC5AC- and MUC5B-expressing cells and augmented MCP-1 expression is associated with increased MUC5AC and MUC5B immunolabeling, suggesting that the mechanisms described in primary cell cultures in the present study are operative in vivo. Therefore, therapeutic approaches targeting MCP-1/CCR2B may be useful in preventing not only influx of inflammatory cells to the airways but also mucus hypersecretion and goblet cell hyperplasia.

scholarly journals The NMDA receptor activation by d-serine and glycine is controlled by an astrocytic Phgdh-dependent serine shuttle

2019 ◽  
Vol 116 (41) ◽  
pp. 20736-20742 ◽  
Author(s):  
Samah Neame ◽  
Hazem Safory ◽  
Inna Radzishevsky ◽  
Ayelet Touitou ◽  
Francesco Marchesani ◽  
...  
Keyword(s):  
De Novo ◽  
Primary Cultures ◽  
Long Term Potentiation ◽  
Receptor Activation ◽  
In Vivo Microdialysis ◽  
Glutamatergic Neurons ◽  
Nmdar Activation ◽  
Serine Metabolism

Astrocytes express the 3-phosphoglycerate dehydrogenase (Phgdh) enzyme required for the synthesis of l-serine from glucose. Astrocytic l-serine was proposed to regulate NMDAR activity by shuttling to neurons to sustain d-serine production, but this hypothesis remains untested. We now report that inhibition of astrocytic Phgdh suppressed the de novo synthesis of l-and d-serine and reduced the NMDAR synaptic potentials and long-term potentiation (LTP) at the Schaffer collaterals-CA1 synapse. Likewise, enzymatic removal of extracellular l-serine impaired LTP, supporting an l-serine shuttle mechanism between glia and neurons in generating the NMDAR coagonist d-serine. Moreover, deletion of serine racemase (SR) in glutamatergic neurons abrogated d-serine synthesis to the same extent as Phgdh inhibition, suggesting that neurons are the predominant source of the newly synthesized d-serine. We also found that the synaptic NMDAR activation in adult SR-knockout (KO) mice requires Phgdh-derived glycine, despite the sharp decline in the postnatal glycine levels as a result of the emergence of the glycine cleavage system. Unexpectedly, we also discovered that glycine regulates d-serine metabolism by a dual mechanism. The first consists of tonic inhibition of SR by intracellular glycine observed in vitro, primary cultures, and in vivo microdialysis. The second involves a transient glycine-induce d-serine release through the Asc-1 transporter, an effect abolished in Asc-1 KO mice and diminished by deleting SR in glutamatergic neurons. Our observations suggest that glycine is a multifaceted regulator of d-serine metabolism and implicate both d-serine and glycine in mediating NMDAR synaptic activation at the mature hippocampus through a Phgdh-dependent shuttle mechanism.

Vasoactive Intestinal Peptide (VIP) and Pituitary Adenylate Cyclase-activating Polypeptide (PACAP) as Modulators of Both Innate and Adaptive Immunity

2002 ◽  
Vol 13 (3) ◽  
pp. 229-237 ◽  
Author(s):  
Doina Ganea ◽  
Mario Delgado
Keyword(s):  
T Cells ◽  
Nuclear Translocation ◽  
De Novo ◽  
Biological Effects ◽  
Inflammatory Cells ◽  
Th2 Cells ◽  
Long Term Memory ◽  
Specific Receptors ◽  
Anti Inflammatory

The structurally related neuropeptides VIP and PACAP are released within the lymphoid organs following antigenic stimulation, and modulate the function of inflammatory cells through specific receptors. In activated macrophages, VIP and PACAP inhibit the production of pro-inflammatory agents (cytokines, chemokines, and nitric oxide), and stimulate the production of the anti-inflammatory cytokine IL-10. These events are mediated through the VIP/PACAP effects on de novo expression or nuclear translocation of several transcription factors, i.e., NFκB, CREB, c-Jun, JunB, and IRF-1. The in vivo administration of VIP/PACAP results in a similar pattern of cytokine and chemokine modulation, which presumably mediates the protective effect of VIP/PACAP in septic shock. In addition, VIP/PACAP reduce the expression of the co-stimulatory molecules B7.1/B7.2, and the subsequent stimulatory activity of macrophages for T-helper cells. In T-cells expressing specific VIP/PACAP receptors, VIP and PACAP inhibit the expression of FasL through effects on NFκB, NFAT, and Egr2/3. The reduction of FasL expression has several biological consequences: inhibition of antigen-induced cell death in CD4 T-cells, inhibition of the FasL-mediated cytotoxicity of CD8 and CD4 effectors against direct and bystander targets, and promotion of long-term memory Th2 cells, through a positive effect on the survival of Th2, but not Th1, effectors. The various biological effects of VIP and PACAP are discussed within the range of a general anti-inflammatory model.

scholarly journals Massively calcified endosomal death (MCED) of endothelial cells

2014 ◽  
Author(s):  
Larry Weisenthal
Keyword(s):  
Endothelial Cells ◽  
Kinase Inhibitors ◽  
Culture Media ◽  
Vascular Inflammation ◽  
Primary Cultures ◽  
Human Tumor ◽  
Inflammatory Cells ◽  
Different Types ◽  
Human Sera ◽  
Contrast Exposure

We have discovered a novel and specific mechanism of endothelial cell death.We refer to this novel death mechanism as massively calcified endosomal death, or MCED. Exposure of endothelial cells to non-specific toxins or other physical stresses induces death by traditional apoptotic and non-apoptotic mechanisms, common to most different types of cells. In contrast, exposure of endothelial cells (but not other types of nucleated cells) to specific insults, such as oxidized pathogenic lipids (e.g. 7-ketocholesterol) or agents with known anti-angiogenic activity (e.g. bevacizumab, certain tyrosine kinase inhibitors, etc.) triggers cell death via a novel pathway, which involves the formation of massively calcified endosomes, which, in turn, escape from the dying endothelial cells as massively calcified exosomes. These endosomes/exosomes appear capable of provoking an inflammatory response, characterized by physical association of calcified microparticles with inflammatory cells (monocytes, lymphocytes, neutrophils) with resulting increased release of an inflammatory mediator (TNF) into the culture medium. Traditional media for the culture of endothelial cells are profoundly inhibitory to MCED, as are some mammalian sera and many human sera, explaining why MCED had not been previously discovered and reported. The present discovery of MCED was accidental, resulting from work with primary cultures of fresh human tumor cell clusters, which invariably contain microcapillary cells Our culture media are optimized for the tumor cells and not for the endothelial cells and, thus, are permissive of MCED. I propose MCED as the central mechanism underlying both intimal calcification and vascular inflammation in atherosclerosis.

Abstract 642: Specific Protein Kinase C Isoforms Are Critical Mediators of Physiologic Downregulation of Perivascular Tissue Factor

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Laura Sommerville ◽  
Kristen Gorman ◽  
Dougald Monroe ◽  
Maureane Hoffman
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Degradation ◽  
Tissue Factor ◽  
De Novo ◽  
Primary Cultures ◽  
Protein Loss ◽  
Transcriptional Inhibition ◽  
Kinase C

Tissue factor (TF) is a procoagulant and transmembrane receptor for FVII(a) that is upregulated in pathological conditions. We recently demonstrated that perivascular TF is downregulated around angiogenic vessels near a cutaneous wound. The goal of this study was to identify mechanisms that mediate TF loss. Primary cultures of human pericytes express high levels of TF. TF expression was lost during culture with phorbol-12 myristate 13-acetate (PMA) for 8 hours, and was maintained for 24 hours. This model recapitulates the pattern of downregulation observed in vivo . Using qRT-PCR we assessed changes in TF gene expression in response to PMA. TF mRNA decreased 4- and 6-fold at 8 and 12 hours after treatment (p<0.01), and remained 2-fold lower in treated cells after 24 hours (p<0.05). Inhibiting de novo transcription with actinomycin D showed that degradation of TF mRNA was similar in PMA- and vehicle-treated groups (p=ns). Thus, downregulation of TF mRNA occurs primarily through inhibition of its synthesis. We next identified a physiologic mediator of TF downregulation using specific inhibitors against PMA-responsive signaling proteins. Two different inhibitors against Protein Kinase C (PKC) were used: Go6983, which inhibits isoforms α, β, δ, ε, μ, and ζ, and GFX, which inhibits α, β, ε, and γ. Both inhibitors significantly attenuated PMA-mediated transcriptional downregulation (p<0.001). Based on overlap of inhibited isoforms, this suggests a minimal role for ζ, μ, δ, and γ, while one or more of the α, β, and ε isoforms appear to be critical mediators of TF mRNA synthesis inhibition. Since the timing of TF protein loss is not fully explained by transcriptional inhibition, we investigated the role of protein degradation in TF loss. When protein synthesis was inhibited by cyclohexamide, addition of PMA shortened the half-life of pericyte TF from 11 hours to 5 hours (p<0.001). This indicates that increased protein degradation contributes to PMA-induced loss of TF expression. Both Go6983 and GFX significantly attenuated TF protein degradation also. Taken together, our data show that TF downregulation is mediated by transcriptional inhibition and protein degradation, and PKC α, β, and ε have emerged as potential mediators of both mechanisms.

scholarly journals Addition of High Molecular Weight Hyaluronic Acid to Fibroblast-Like Stromal Cells Modulates Endogenous Hyaluronic Acid Metabolism and Enhances Proteolytic Processing and Secretion of Versican

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1681
Author(s):  
Jiapeng Xue ◽  
Jinnan Chen ◽  
Quan Shen ◽  
Deva Chan ◽  
Jun Li ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Hyaluronic Acid ◽  
High Molecular Weight ◽  
Stromal Cells ◽  
De Novo ◽  
Linear Chain ◽  
Primary Cultures ◽  
Culture Conditions ◽  
Proteolytic Processing

We have examined the effect of exogenous linear chain high molecular weight hyaluronic acid (HMW HA) on endogenously synthesized hyaluronic acid (HA) and associated binding proteins in primary cultures of fibroblast-like stromal cells that were obtained by collagenase digestion of the murine peripatellar fat pad. The cultures were expanded in DMEM that was supplemented with fetal bovine serum and basic fibroblast growth factor (bFGF) then exposed to macrophage-colony-stimulating factor (MCSF) to induce macrophage properties, before activation of inflammatory pathways using E. coli lipopolysaccharide (LPS). Under all culture conditions, a significant amount of endogenously synthesized HA localized in LAMP1-positive lysosomal vesicles. However, this intracellular pool was depleted after the addition of exogenous HMW HA and was accompanied by enhanced proteolytic processing and secretion of de novo synthesized versican, much of which was associated with endosomal compartments. No changes were detected in synthesis, secretion, or proteolytic processing of aggrecan or lubricin (PRG4). The addition of HMW HA also modulated a range of LPS-affected genes in the TLR signaling and phagocytosis pathways, as well as endogenous HA metabolism genes, such as Has1, Hyal1, Hyal2, and Tmem2. However, there was no evidence for association of endogenous or exogenous HMW HA with cell surface CD44, TLR2 or TLR4 protein, suggesting that its physiochemical effects on pericelluar pH and/or ionic strength might be the primary modulators of signal transduction and vesicular trafficking by this cell type. We discuss the implications of these findings in terms of a potential in vivo effect of therapeutically applied HMW HA on the modification of osteoarthritis-related joint pathologies, such as pro-inflammatory and degradative responses of multipotent mesenchymal cells residing in the synovial membrane, the underlying adipose tissue, and the articular cartilage surface.

scholarly journals De Novo Expression of Dopamine D2 Receptors on Microglia after Stroke

2015 ◽  
Vol 35 (11) ◽  
pp. 1804-1811 ◽  
Author(s):  
Jojanneke HJ Huck ◽  
Dorette Freyer ◽  
Chotima Böttcher ◽  
Mihovil Mladinov ◽  
Claudia Muselmann-Genschow ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
De Novo ◽  
D2 Receptor ◽  
Inflammatory Cells ◽  
Striatal Neurons ◽  
Infarct Core ◽  
Ischemic Brain ◽  
Murine Microglia ◽  
Green Fluorescent

Dopamine is the predominant catecholamine in the brain and functions as a neurotransmitter. Dopamine is also a potent immune modulator. In this study, we have characterized the expression of dopamine receptors on murine microglia. We found that cultured primary microglia express dopamine D1, D2, D3, D4, and D5 receptors. We specifically focused on the D2 receptor (D2R), a major target of antipsychotic drugs. Whereas D2Rs were strongly expressed on striatal neurons in vivo, we did not detect any D2R expression on resident microglia in the healthy brains of wild-type mice or transgenic mice expressing the green fluorescent protein (GFP) under the control of the Drd2 promoter. However, cerebral ischemia induced the expression of D2R on Iba1-immunoreactive inflammatory cells in the infarct core and penumbra. Notably, D2R expression was confined to CD45hi cells, and GFP BM chimeras revealed that D2R was expressed on activated resident microglia as well as on peripherally derived macrophages in the ischemic brain. Importantly, the D2/3R agonist, pramipexole, enhanced the secretion of nitrite by cultured microglia in response to proinflammatory stimuli. Thus, dopamine may serve as a modulator of microglia function during neuroinflammation.

Radiolabeled r-Hirudin as a Measure of Thrombin Activity at, or within, the Rabbit Aorta Wall In Vitro and In Vivo

1994 ◽  
Vol 71 (04) ◽  
pp. 499-506 ◽  
Author(s):  
Mark W C Hatton ◽  
Bonnie Ross-Ouellet
Keyword(s):  
Rabbit Aorta ◽  
Balloon Injury ◽  
Recombinant Hirudin ◽  
Aorta Wall ◽  
Thrombin Activity ◽  
Before And After ◽  
The Matrix

SummaryThe behavior of 125I-labeled recombinant hirudin towards the uninjured and de-endothelialized rabbit aorta wall has been studied in vitro and in vivo to determine its usefulness as an indicator of thrombin activity associated with the aorta wall. Thrombin adsorbed to either sulfopropyl-Sephadex or heparin-Sepharose bound >95% of 125I-r-hirudin and the complex remained bound to the matrix. Binding of 125I-r-hirudin to the exposed aorta subendothelium (intima-media) in vitro was increased substantially if the tissue was pre-treated with thrombin; the quantity of l25I-r-hirudin bound to the de-endothelialized intima-media (i.e. balloon-injured in vitro) correlated positively with the quantity of bound 131I-thrombin (p <0.01). Aortas balloon-injured in vivo were measured for thrombin release from, and binding of 125I-r-hirudin to, the de-endothelialized intimal surface in vitro; 125I-r-hirudin binding correlated with the amount of active thrombin released (p <0.001). Uptake of 125I-r-hirudin by the aorta wall in vivo was proportional to the uptake of 131I-fibrinogen (as an indicator of thrombin activity) before and after balloon injury. After 30 min in the circulation, specific 125I-r-hirudin binding to the uninjured and de-endo- thelialized (at 1.5 h after injury) aorta wall was equivalent to 3.4 (± 2.5) and 25.6 (±18.1) fmol of thrombin/cm2 of intima-media, respectively. Possibly, only hirudin-accessible, glycosaminoglycan-bound thrombin is measured in this way.

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